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THE ROLES OF METALLOTHIONEIN EXPRESSION AND DIETARY ZINC IN

ZINC METABOLISM AND CYTOPROTECTION IN METALLOTHIONEIN

TRANSGENIC AND METALLOTHIONEIN KNOCKOUT MICE

By
STEVEN ROGER DAVIS

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2000

This work is dedicated to the memory of my aunt, Leona Goguen. She has been, and will
always be my role model. I dream that one day, I too will possess the wisdom, character,
and courage that she displayed each day of her life.

ACKNOWLEDGMENTS

I would like to thank my committee chairman, Dr. Robert J. Cousins for his
guidance throughout this work.

I would like to thank my committee members Dr. Jesse Gregory, Dr. Rachel
Shireman, Dr. Susan Percival, and Dr. Stephen Roberts for their advice and input into
this work.

I would like to thank Walter Jones, Virginia Mauldin and Warren Clark for their
technical assistance and sense of humor throughout my stay in Dr. Cousins' laboratory.

I would like to thank Dr. Nora Holquist, Dr. Christina Khoo, Dr. Barbara Davis,
Dr. Vicki Sullivan, Dr. Lorraine Lanningham-Foster, Dr. Jay Cao, Monique Coy,
Jennifer Moore and Juan Liuzzi for their friendship and support.

I would like to thank Dr. Raymond Blanchard and Dr. Robert McMahon for their
friendship and tutoring in the laboratory.

I would like to thank my family for their patience during this process.

Lastly, I would like to thank Amy Mackey for her technical and professional
assistance and especially for her personal support during the final stages of this work.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS Hi

ABBREVIATIONS vi

ABSTRACT vii

CHAPTERS

1 INTRODUCTION 1

Literature Review 2

Hypotheses and Research Objectives 9

2 THE EFFECT OF METALLOTHIONEIN EXPRESSION ON ZINC

ABSORPTION IN METALLOTHIONEIN TRANSGENIC AND

METALLOTHIONEIN KNOCKOUT MICE 11

Introduction 11

Materials and Methods 12

Results 15

Discussion 21

3 REGULATION OF METALLOTHIONEIN EXPRESSION

AND ZINC METABOLISM BY DIETARY ZINC IN METALLOTHIONEIN

TRANSGENIC AND METALLOTHIONEIN KNOCKOUT MICE 28

Introduction 28

Materials and Methods 33

Results 32

Discussion 36

4 THE EFFECTS OF METALLOTHIONEIN GENE EXPRESSION AND

SUPPLEMENTAL DIETARY ZINC IN PROTECTION AGAINST
HEPATOTOXICITY IN METALLOTHIONEIN TRANSGENIC AND

METALLOTHIONEIN KNOCKOUT MICE 41

Introduction 41

Materials and Methods 43

Results 47

Discussion 56

5 EFFECTS OF METALLOTHIONEIN GENE EXPRESSION AND

SUPPLEMENTAL ZINC IN PROTECTION AGAINST OXIDATIVE
STRESS IN PRIMARY HEPATOCYTE CULTURES FROM
METALLOTHIONEIN TRANSGENIC AND METALLOTHIONEIN

KNOCKOUT MICE 66

Introduction 66

Materials and Methods 67

Results 72

Discussion 76

6 SUMMARY AND CONCLUSIONS 85

Zinc and Metallothionein in Zinc Absorption and Metabolism 85

Zinc and Metallothionein in Defense Against Oxidative Stress 89

LITERATURE CITED 94

BIOGRAPHICAL SKETCH 109

ABBREVIATIONS

AAS

ALT

BBM

Dex

DTNB

GSH

GSSG

i.p.

II- 1

11-6

LDH

LPS

MRE

MTT

NPT

SPAEC

SSA

TBH

TCA

WME

atomic absorption spectrophotometry

alanine aminotransferase enzyme

brush border membrane

control mouse strain for KO mice

corn oil

control mouse strain for TG mice

dexamethasone

5,5'-dithio-bis(2-nitrobenzoic acid)

reduced glutathione

oxidized glutathione

intraperitoneal

interleukin- 1

interleukin-6

metallothionein-null (knockout) mouse

lactate dehydrogenase enzyme

lipopolysaccharide

metal response element

metallothionein

thiazoyl blue

nonprotein thiol

perivenous

sheep pulmonary artery endothelial cell

sulfosalicylic acid

tertiary-butyl hydroperoxide

trichloroacetic acid

metallothionein overexpressing transgenic mouse

total thiol

William's medium E

Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Master of Arts

THE ROLES OF METALLOTHIONEIN EXPRESSION AND DIETARY ZINC IN

ZINC METABOLISM AND CYTOPROTECTION IN METALLOTHIONEIN

TRANSGENIC AND METALLOTHIONEIN KNOCKOUT MICE

Steven Roger Davis

December 2000

Chairman: Robert J. Cousins

Major Department: Food Science and Human Nutrition Department

The biochemistries of zinc and metallothionein are intricately linked. Zinc

induces metallothionein gene expression and allows the protein to resist proteolysis. In

turn, binding of zinc to metallothionein allows cellular zinc accumulation, and

metallothionein may be involved in intracellular zinc trafficking. Further, metallothionein

induction and zinc accumulation often are coupled. This relationship makes it difficult to

determine if effects of zinc supplementation are due to zinc, metallothionein induction, or

both. Metallothionein overexpressing (transgenic) mice and metallothionein null

(knockout) mice provide unique models to study the effects of zinc supplementation and

metallothionein gene expression on physiological processes. We used these mouse

models to determine (1) the effects of metallothionein expression and dietary zinc intake

on zinc absorption and tissue zinc accumulation, and (2) the effects of zinc

supplementation and/or metal lothionein gene expression on susceptibility to oxidative
stress.

Metallothionein expression was inversely proportional to serum zinc
concentrations 2 h after an oral zinc dose, which strengthens the theory that
metallothionein impedes zinc absorption. Intestinal zinc accumulation was inversely
related to metallothionein expression, however, which argues that metallothioneinein
does not act by simply sequestering zinc in the intestine. Metallothionein protein
expression was directly proportional to intestinal and liver zinc concentrations after 3-7 d
of zinc supplementation, but only at dietary zinc levels 20- to 50-fold of the requirement.
Intestine and liver zinc concentrations did not change over a wide range of zinc intakes,
and knockout mice maintained serum zinc levels as well as control mice. These points
suggest that at typical zinc intakes, and in the absence of significant stresses,
maintenance of zinc homeostasis is not metallothionein-dependent.

Metallothionein expression protected against carbon tetrachloride-induced
hepatotoxicity. Neither zinc supplementation nor metallothionein overexpression
provided further protection, however. The heightened toxicity in KO mice after carbon
tetrachloride treatment was associated with their lack of control over zinc homeostasis. In
primary hepatocyte cultures, metallothionein induction was associated with increased
susceptibility to oxidative stress. This was likely due to the observed depression of
cellular glutathione. These results argue against direct antioxidant roles for
metallothionein expression and supplemental zinc in mouse liver.

Vlll

CHAPTER 1
INTRODUCTION

The objectives of the research reported in this dissertation were to determine (1)
the roles that dietary zinc and metallothionein have in regulation of zinc absorption and
tissue distribution, and (2) the roles that dietary zinc and metallothionein have in defense
against oxidative stress. The production of mouse strains with perturbed metallothionein
expression provided the opportunity to investigate the above objectives under conditions
of metallothionein absence and metallothionein excess in an intact animal model.
Previously, investigators studying the effects of metallothionein expression on zinc
metabolism or oxidative stress used a myriad of treatments to alter metallothionein levels
before their experiments. Although they successfully altered metallothionein levels, other
physiological pathways may have been perturbed as well. For example, zinc pretreatment
induces metallothionein, but undoubtedly affects other components of the machinery that
regulates zinc metabolism (e.g., zinc transporter abundance). Similarly, treatment with
other metals, hormones, and cytokines is known to affect more than metallothionein. By
using mice with altered metallothionein expression the complications associated with
such treatments were avoided. Because of the artificial nature of these models, we must
use caution when interpreting the results of studies in which they were used.
Nevertheless, the results of such studies provided strong support for existing theories, as
well as new insights for putative roles for metallothionein.

Literature Review
Zinc

Zinc is an essential nutrient whose recommended dietary allowance (RDA; 15
mg/d for men, 12 mg/d for women) ranks with iron as highest among the trace elements
(National Research Council 1989). Zinc functions in more than 50 enzymes, serving in
catalytic, structural, and regulatory roles (reviewed by Vallee and Falchuck 1993,
Cousins 1996). These enzymes are involved in the synthetic and catabolic pathways of
many biomolecules, including proteins, nucleic acids, carbohydrates and lipids. Zinc also
is a component of zinc finger transcription factors. Zinc is distributed throughout the
body, including 57% residing in skeletal muscle, followed by 29% in bone, 6% in skin,
and 5% in liver (reviewed by Jackson 1989). Within cells zinc is distributed ubiquitously.
Thiers and Vallee (1957) reported that 43% of liver zinc is cytosolic, 37% nuclear, 13%
microsomal, 5% mitochondrial, and 2% in connective tissue. Zinc deficiency is
associated with a 20% reduction in whole body zinc, but it is unknown which
intracellular pool(s) is most affected (Hambidge 1989). Although severe zinc deficiency
is uncommon, mild zinc deficiency may be prevalent in many parts of the world (Prasad
1991).

Metallothionein

Metallothioneins are a family of small (6-7 kDa), cysteine-rich metal binding
proteins found in vertebrates and invertebrates (Dunn et al. 1987). These proteins are
further characterized by a lack of histidine residues, aromatic amino acids, and disulfide
bonds. The protein is capable of binding up to (10) copper atoms, or up to (7) cadmium
and/or zinc atoms in two distinct clusters of the protein (Neilson et al. 1985).

Metallothioneins are thought to function in metal homeostasis, including zinc absorption
and tissue distribution, and protection against heavy metal toxicity (Cousins 1985, Liu et
al. 1995, Masters et al. 1994A). Metallothioneins are transcriptionally regulated by
metals through metal response elements (MREs) in their gene promoters (Carter et al.
1984, Durnam and Palmiter 1981, Stuart et al. 1984). Metallothioneins also are
transcriptionally regulated by glucocorticoid hormones and cytokines, which implies a
role for metallothioneins in inflammatory and stress-related responses, such as the acute
phase response (Cousins and Leinart 1988, Etzel et al. 1979).

Although four forms of metallothionein have been discovered, the two most
widely expressed are metallothionein- 1 and metallothionein-2 (Hunziker et al. 1995,
Kagi et al. 1974). These forms are found in most tissues, and are especially prevalent in
the liver, kidney, pancreas, and intestine (Hunziker and Kagi 1985). Metallothionein-3
and metallothionein-4 forms were discovered recently, and are expressed mostly in the
brain and skin, respectively (Masters et al. 1994B, Quaife et al. 1994). Subforms of
metallothionein (e.g., MT-la) exist in primates, but not in mice (Stennard et al. 1994,
Twunoo et al. 1978). Because metallothionein- 1 and metallothionein-2 are the
predominant forms in the liver and the intestine, they are the focus of discussion from
this point on.

Metallothionein in Zinc Absorption and Tissue Distribution

Chapters 2 and 3 of this dissertation focus on the role of metallothionein in
dietary zinc absorption and tissue zinc accumulation. The quantity of zinc absorbed by
the body depends on several processes. These include the ingestion and digestion of zinc-
containing foodstuffs, uptake of zinc by the intestinal mucosa, and transport of zinc from

the intestine to the vascular supply. Zinc uptake by the intestine, followed by transfer to
the portal blood supply is referred to collectively as zinc absorption. Zinc is absorbed
throughout the entire small intestine (Lee et al. 1989). The predominant site in humans is
the jejunum, while both the duodenum and jejunum appear to be major sites in rodents
(Davies 1980, Lee et al. 1989). Although some information is known about zinc
absorption, a clear understanding of this process and its regulation has not been reached.
The process can be divided into three parts: (1) uptake from the lumen at the brush
border membrane (BBM), (2) transport across the epithelial cell, and (3) transfer of zinc
across the basolateral membrane to the vascular bed. Zinc absorption is dependent on the
concentration of bioavailable zinc in the lumen. Although ~ 30% of zinc is absorbed
from typical diets, greater efficiency is achieved in animals fed zinc-deficient diets, and
lesser efficiency in animals fed diets containing supplemental zinc (Hempe and Cousins
1992, Sandstrom 1989, Smith et al. 1978). In vitro transport studies using brush border
membrane vesicles from rats showed that BBM transport was greater from zinc-deficient
rats compared to zinc-adequate rats (Menard and Cousins 1983). Dietary zinc intake did
not affect transport into basolateral membrane vesicles, however, suggesting that control
of zinc absorption occurs at the apical, but not the basolateral membrane (Oestreicher and
Cousins 1989).

The last area for control of zinc absorption is the enterocyte cytosol itself. The
cytosolic protein metallothionein has been the focus of research in this area because it is
induced by high dietary zinc and parenteral zinc administration, but is depressed at low
zinc intakes (Menard and Cousins 1983, Smith et al. 1978). One model of zinc absorption
suggests that intestinal metallothionein is an integral part of the regulatory machinery,

acting as a damping agent during periods of excessively high dietary zinc intakes
(Cousins 1989, Hoadley et al. 1988, Richards and Cousins 1975). In this model a high
zinc influx into the mucosal cell induces metallothionein production. Metallothionein
then chelates the cytosolic zinc, limiting its passage from the enterocyte to the portal
circulation. Enterocyte to lumen efflux of zinc, combined with regular sloughing of
enterocytes from the villus tip result in reduced zinc absorption. This model is consistent
with results from animal studies wherein high zinc diets or parenteral zinc administration
elevated metallothionein levels in the intestine and resulted in decreased zinc absorption
from subsequent meals (Coppen and Davies 1987, Hoadley et al. 1988, Richards and
Cousins 1975). Similarly dietary zinc restriction depresses tissue metallothionein content
and results in enhanced zinc absorption from subsequent meals (Hoadley et al. 1987,
Smith and Cousins 1980).

Contrary to results with diet-related changes in metallothionein, several inducers
of intestinal metallothionein do not inhibit zinc absorption. For instance, bacterial
infection, bacterial lipopolysaccharide injection, and interleukin- 1 administration each
increased zinc absorption, even though each induces intestinal metallothionein (Pekarek
and Evans 1975, Pekarek and Evans 1976). Further, mouse studies showed no clear
correlation between metallothionein level of the intestine and zinc absorption using either
oral dosing, stomach tube feeding, in situ duodenal loop feeding, or injection of a zinc
solution into the duodenum in situ (Flanagan et al. 1983, Olafson 1983, Starcher et al.
1980). Other studies have shown no increase in intestinal zinc retention even when
metallothionein is induced (Hempe et al. 1991). Thus the role of metallothionein in
regulation or zinc absorption is still in question.

Metallothionein also is believed to affect zinc accumulation in tissues.
Metallothionein gene expression is regulated in a tissue-specific manner in rats (Blalock
et al. 1988). Hepatic metallothionein induction occurs after excessive dietary zinc intakes
in rats, and is associated with hepatic zinc accumulation (McCormick et al. 1981). Other
treatments that induce hepatic metallothionein, such as administration of interleukin- 1 ,
interleukin-6, and lipopolysaccharide, result in hepatic zinc accumulation and serum zinc
depression (De et al. 1990, Huber and Cousins 1993). Taken together, these data suggest
that metallothionein expression exerts a strong influence on tissue zinc distribution.

Within cells, metallothionein also may function as a zinc reservoir from which
this metal is made available for incorporation into apometalloenzymes or other
metalloproteins. This idea is supported by the highly rapid exchange rates of zinc in
metallothionein, which are far faster than the exchange rates from other proteins (Li et al.
1980, Udom and Brady 1980). Further, incubation with Zn-metallothionein reconstitutes
a number of enzymes and transcription factors (e.g., apocarbonic anhydrase and Spl) and
rescues their activities (Li et al. 1980, Udom and Brady 1980, Zeng et al. 1991).
Exchange rates are more rapid and more zinc is exchanged in the presence of oxidized
glutathione, possibly linking zinc release to cellular redox status (Jiang et al. 1998).
Interestingly, zinc is also liberated from metallothionein by a number of oxidants
(Berendji et al. 1997, Fliss and Menard 1992).

We undertook research concerning the role of metallothionein in regulation of
zinc absorption due to the importance of zinc for health. Proper zinc nutrition is
particularly important to support growth and immune functions (reviewed in Keen and
Gershwin 1990, Rivera et al. 1995). In particular, zinc deficiency is known to retard

growth and inhibit sexual maturity, both of which can be reversed to some degree with
zinc supplementation (as reviewed in Prasad 1991). Further, supplemental zinc can help
to alleviate secretory diarrhea and morbidity in third world countries (Sazawal et al.
1995). Consequently, determining the mechanism(s) and regulation of zinc absorption
provides information that can be used to efficiently and effectively maintain proper zinc
homeostasis, and to support health.

Metallothionein and Zinc in Defense Against Oxidative Stress

The pathogenesis of aging, cancer, atherosclerosis, cataracts, neurodegenerative
disorders, and ischemia-reperfusion injury are associated with oxidative stress (Blot et al.
1993, Weidau-Pazos et al. 1996, Fraga et al. 1990, Rengstrom et al. 1992, Rosen et al.
1993, Taylor et al. 1992). Oxidative stress occurs when the balance between oxidative
attacks and oxidative defense systems favors oxidation. The mediators of oxidative
damage include reactive oxygen species (i.e., H202, -02 and OH), and radical species that
are not oxygen-centered (e.g., *CC13). These species can cause damage to lipids, proteins,
and nucleic acids (Farber 1994, Loft et al. 1994, Oliver et al. 1990). Living organisms
combat oxidative stress through the use of antioxidant nutrients such as tocopherols and
ascorbate, as well as endogenous antioxidant scavengers like superoxide dismutase and
glutathione (Yu 1994). When damage does occur, organisms have damage repair systems
to fall back on. For example, DNA excision repair systems remove oxidized DNA bases,
and glutathione peroxidase can convert lipid hydroperoxides formed by membrane
oxidation to less reactive lipid hydroxides. Further, proteolytic and lipolytic enzymes
degrade damaged macromolecules when damage is irreversible. But when these systems
are overcome, the cell or organism may not survive. Since the generation of reactive

oxygen species is unavoidable, maximizing antioxidant defense systems has become a
research priority (Cohen 1994).

Adequate zinc nutrition may help protect against oxidative stress. Zinc-deficient
rodents display markers of oxidative damage, and are more susceptible to subsequent
oxidative stresses (DiSilvestro and Carlson 1993, Miceli et al. 1999, Oteiza and Keen
1995). These results may reflect depressed activity of Cu/Zn superoxide dismutase,
increased oxidation of sulfydryl groups that are normally protected by zinc binding, or
increased exposure of zinc binding sites within proteins to copper- and iron-induced
oxidation (as reviewed in Powell 2000). Increased oxidative stress also might be due to
perturbation of a number of other zinc-dependent processes, including maintenance of
cellular metallothionein (Blalock et al. 1988, Schroeder and Cousins 1990). This is due to
reduced activation of metallothionein gene expression, as well as enhanced susceptibility
of metallothionein protein to proteolysis when zinc is not available for binding (Smith et
al. 1978, Feldman and Cousins 1976). Metallothionein expression may help defend
against oxidative stress since metallothionein is capable of scavenging free radicals, and
the hydroxyl radical in particular (Thornalley and Vasak 1985).

Beyond preventing zinc deficiency, supplemental zinc provides additional
protection against certain oxidative stresses (Blain et al. 1998, Dhawan and Goel 1995).
Although the mechanism of protection is uncertain, it may include the induction of
metallothionein protein. Metallothionein is induced by a number of chemicals that
generate oxidative stress (Bauman et al. 1991, Satoh et al. 1996, Shiraishi et al. 1989;
Tate et al. 1995). Preinduction of metallothionein by a number of agents (including zinc,
other metals, hormones, and cytokines) is associated with protection against the toxicity

of subsequent metal, chemical, and other stresses in cell culture and in vivo (e.g., Coppen
et al. 1988, Moffat et al. 1996, Satoh et al. 1992, Schroeder and Cousins 1990). Similar
results were seen when cells were transfected with metallothionein genes (Kaina et al.
1990, Schwarz et al 1995). Other reports showed no protection by metallothionein
expression against free radicals, however, casting doubt on metallothionein' s role in
oxidative defense (Kaina et al. 1990, Kelley et al. 1988).

Based on these observations, supplemental zinc and metallothionein expression
may or may not affect the outcome of conditions characterized by oxidative stress
(Oteiza et al. 1995, Prasad 1991). Characterization of zinc and metallothionein as
participants in oxidative defense is important because oxidative damage is associated
with so many disease processes. For example, oxidative stress is a component of
inflammatory bowel disease, which is associated with a reduced metallothionein content
of the bowel (Mulder et al. 1991). Also, free radical production and oxidative stress
characterize many diseases of the liver, and the liver is an organ that contains high zinc
and metallothionein concentrations after zinc supplementation (Cohen 1994).

Hypotheses and Research Objectives

Based on the information summarized above, we developed two hypotheses
regarding the biochemical actions and interactions of zinc and metallothionein:

1 . Zinc absorption is inversely related to intestinal metallothionein production,
while tissue distribution of absorbed zinc is directly related to the
metallothionein content of the tissue.

2. Zinc and/or metallothionein protect mouse livers and hepatocyte cultures from
oxidative damage.

We examined these hypotheses using relatively new models for biological
research - transgenic mice. We used metallothionein transgenic overexpressing mice
(designated TG), metallothionein knockout mice (i.e., null; designated KO), and their
respective control strains in all of the studies that follow. Metallothionein transgenic mice
carry 56 copies of the metallothionein- 1 gene in their genome, and those genes are
responsive to the same stimuli that induce endogenous metallothionein- 1 genes (Palmiter
et al. 1993). In contrast, metallothionein knockout mice produce no metallothionein- 1 or
-2 protein under any conditions in the tissues we studied (Masters et al. 1994A). This
allows us to assess the effects of a broad spectrum of metallothionein expression levels
on zinc metabolism and oxidative stress. Further, studies with metallothionein null mice
allow determination of biological effects of zinc that are independent of metallothionein.

CHAPTER 2

THE EFFECT OF METALLOTHIONEIN EXPRESSION ON ZINC ABSORPTION IN

METALLOTHIONEIN TRANSGENIC AND METALLOTHIONEIN KNOCKOUT

MICE

Introduction

The mechanisms that regulate zinc metabolism are not understood. When dietary
zinc intake is restricted in experimental animals and humans, the efficiency of zinc
absorption increases and endogenous zinc excretion decreases. Furthermore, zinc
absorption is depressed after ingestion of zinc-rich diets. The biomolecules that mediate
the regulation of zinc metabolism by the dietary zinc supply have not been fully
described. The cytosolic protein metallothionein (MT) may be a principal participant in
this regulation. This metalloprotein is inducible by many factors (stimuli) and may act as
a zinc pool or buffer that is influenced by body zinc levels. In addition, the redistribution
of endogenous zinc associated with stresses such as acute infection and physical trauma
may require metallothionein. The induction is believed to involve interleukin-1,
interleukin-6, and glucocorticoid hormone-mediated changes, all of which can be linked
to elevated expression of metallothionein in the liver and other tissues (reviewed in
Cousins 1989, 1996).

Metallothionein transgenic mice (TG) and metallothionein knockout mice (KO)
provide a unique model to study the effects of metallothionein expression on zinc
absorption. Metallothionein transgenic mice have elevated metallothionein protein in
many tissues, including the liver and intestine (Iszard et al. 1995, Liu and Klaassen

1996). The larger cytosolic metallothionein pools might convey protection against zinc
deficiency (Dalton et al. 1996). Conversely, metallothionein knockout mice allow
examination of how zinc metabolism differs when no metallothionein is produced
(Masters et al. 1994, Michalska and Choo 1993). Others have shown that KO mice have
altered zinc metabolism, including inability to sequester zinc in the liver after injections
of zinc or lipopolysaccharide (Coyle et al. 1995, Philcox et al. 1995). Further,
hepatocytes from KO mice were incapable of accumulating zinc in response to
interleukin-6 or dexamethasone treatment. Hence, significant perturbations of zinc
metabolism occur in mice with altered metallothionein expression. This study was
directed at determining the effects of altered metallothionein expression on zinc
absorption.

The intestine is a major control site for zinc homeostasis and is also a major
metallothionein-expressing organ (Cousins 1989). While some data support an inverse
relationship between intestinal metallothionein expression and zinc absorption (Hoadley
et al. 1987, Hoadley et al. 1988, Menard et al. 1981, Smith and Cousins 1980), other data
do not (Flanagan et al. 1983, Starcher et al. 1980). To examine how metallothionein
influences the intestinal processing of zinc, a zinc dose was delivered by gavage (zinc
tolerance test), and the increase in the serum zinc concentration was used as a measure of
absorption.

Materials and Methods
Animals

The founder mice used in this study were obtained from The Jackson Laboratory,
Bar Harbor, ME. The transgenic mice (designated TG mice) were derived from the

C57BL/6 strain crossed with the SJL strain (Palmiter et al. 1993). The metallothionein
knockout mice (designated KO mice) were derived from the 129/SvCPJ strain crossed
with C57BL/6 (Masters et al. 1994). Mice of the appropriate background strains served
as controls, designated CT and CK, respectively. Only adult male mice were used for
experiments. They were housed in plastic cages with wood shavings as bedding and with
a 12 h light/dark cycle. The mice had free access to a commercial diet (Laboratory
Rodent Diet No. 5001, PMI Feeds, New Albany, IN). All experiments were started
between 8:00 and 10:00 AM. Care and treatment of the mice received approval of the
University of Florida Institutional Animal Care and Use Committee.

Radioisotopes

The a32P-dCTP was from Du Pont NEN (Boston, MA), and the 109Cd (1.35 x 106
kBq/nmol Cd) was from Isotope Product Laboratories (Burbank, CA).

RNA Isolation and Northern Analysis

Total RNA was isolated from intestine and liver using TRIzol reagent (Life
Technologies, Gaithersburg, MD). Briefly, 50 to 100 mg of the proximal duodenum and
the liver were homogenized in 2 mL of TRIzol reagent. After addition of chloroform, the
RNA was processed and analyzed as described previously (Blanchard and Cousins 1996).
Equal quantities of RNA from mice of each group were pooled and subjected to Northern
analysis (20 ug total RNA per lane). Equal loading was confirmed by ethidium bromide
staining. Northern blot analyses were carried out using a rat metallothionein- 1 cDNA
probe (Blanchard and Cousins 1996). It was radiolabeled with a32P-dCTP using the RTS
RadPrime DNA Labeling System (Life Technologies) as described previously

(Blanchard and Cousins 1996). The metallothionein cDNA probe hybridizes to
metallothionein mRNA from the control mice and the KO mice. The metallothionein
mRNA of the KO mice contains a premature stop codon, however, and can not be
translated into MT protein.

High Performance Liquid Chromatography of Intestinal Cytosol

In some experiments, the mucosa was homogenized with a Potter Elvejhem tissue
grinder using 2 volumes of ice-cold buffer (S-12 buffer, 154 mmol/L NaCl, 10 mmol/L
TrisCl, 3 mmol/L NaN3, and 10 mmol/L MgS04) plus protease inhibitors (0.1 mmol/L
phenylmethylsulfonyl fluoride, 1.2 umol/L leupeptin and 1.5 umol/L pepstatin A). After
centrifugation at 100,000 x g (30 min), the cytosol fraction was filtered (0.22 urn) and
200 uL was applied to two Superdex 75 chromatography columns (1 x 30 cm; Pharmacia
Biotech, Piscataway, NJ) in series using an isocratic elution of S-12 buffer (Hempe and
Cousins 1989). Metallothionein was measured in all fractions using the cadmium (109Cd)-
binding assay (Eaton and Toal 1982).

Oral Zinc Dosing

Mice that had been fasted overnight were administered 0.5 mmol Zn/kg body
weight as ZnS04 in saline, or saline alone via a stomach tube and were killed 2 h later.
Blood was obtained by cardiac puncture and serum was prepared for serum zinc analysis.
Liver and intestinal zinc concentrations were measured to determine if the zinc dose
produced a change in tissue uptake/retention.

Analytical Methods and Statistical Analysis

109Cd was measured using a Packard Cobra II gamma spectrometer equipped with
a 3 inch crystal (Packard, Downers Grove, IL). Metallothionein protein was measured by
the cadmium (109Cd) binding assay (Eaton and Toal 1982). Briefly, tissue extracts are
boiled, and the resulting supernatant is incubated with 109Cd. After removal of unbound
109Cd using hemoglobin as a chelator, 109Cd bound to MT is measured by y-counting, and
converted to moles of MT using the Cd-MT binding stoichiometry of 7: 1. Total protein
was measured by the method of Lowry et al. (1951). Serum zinc concentrations were
measured by flame atomic absorption spectrophotometry (AAS) (Hempe and Cousins
1989). Tissue zinc was also measured by AAS, after sections of liver and intestine were
digested with acid (HN03/H2S04; 3/1) as described previously (Dunn and Cousins 1989).
Data were analyzed by ANOVA followed by the Student-Newman-Keuls multiple
comparison test where appropriate (InStat, GraphPad, San Diego, CA). Logarithmic
transformation of some data was used to obtain homogenous variances before analysis.

Results
Metallothionein Expression in Intestine and Liver after Oral Dosing

Northern analysis of metallothionein mRNA in liver and intestine demonstrates
both basal and zinc-induced levels of expression in these genotypes (Fig. 2-1). All groups
expressed metallothionein mRNA, but TG mice had several-fold greater metallothionein
expression than either of the control strains in both the intestine and liver.Metallothionein
mRNA was also expressed in KO mice, but this message contains a premature stop
codon and is not translatable. Zinc treatment resulted in induction of metallothionein
mRNA in the intestine and liver of all mice, but expression was greatest by far in the TG

mice. These data confirm that KO mice, control mice (CK and CT), and TG mice
represent groups with distinguishably different basal and zinc-induced metallothionein
mRNA levels in both intestine and liver. Consequently, if metallothionein is important in
regulating zinc absorption, these groups should display different absorption
characteristics.

The knockout mutation was confirmed by identifying metallothionein protein in
size exclusion chromatography fractions of intestinal cytosol (Fig. 2-2). Metallothionein
protein was measured in each 0.5 mL fraction using the 109Cd binding assay (Eaton and
Toal 1982). A large metallothionein peak is seen between 32 and 35 mL in the
chromatography profile from TG mice, but none is identifiable in the profile from the KO
mice. No peak was seen in profiles from zinc treated KO mice (data not shown).
Therefore, although metallothionein mRNA is produced by KO mice, no metallothionein

+ + +

Zinc

Intestine

Liver

Figure 2-1. Intestinal metallothionein mRNA in metallothionein knockout (KO),
knockout control (CK), transgenic (TG), and transgenic control (CT) mice that
consumed diets containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. Equal
amounts of total RNA were pooled from intestine of 3-5 mice per group and analyzed
by northern analysis using a metallothionein- 1 cDNA probe, with P-Actin used for
normalization. (A) KO and CK mouse intestine. Although metallothionein- 1 mRNA is
present in KO mice, it is not translatable. (B) CT and TG mouse intestine. (C)
Graphical representation of metallothionein- 1 mRNA expression as a function of

protein results from that message. Although metallothionein is the predominant 109Cd
binding compound in the cytosol, a small but finite amount of 109Cd binding is seen in
nearly all other fractions in the profiles of TG and KO mice (Fig. 2-2). Others have
observed the same phenomenon (Liu et al. 1996A). Since no 109Cd binding activity is

18 20 22 24 26 28 30 32 34 36 38 40
Elution Volume (ml_)

Figure 2-2. Superdex 75 size exclusion chromatography of metallothionein (MT) in
cytosol from intestine mucosa of metallothionein-transgenic (TG) and metallothionein-
knockout (KO) mice 2 h after an oral dose of saline. Mucosal cytosol was separated by
two superdex 75 columns run in series and 0.5 mL fractions were collected. The MT
content of fractions 18 through 40 is expressed as fig MT equivalents. Metallothionein
elutes between 32 and 35 mL.

seen in any chromatographic fractions in the KO mice, including the fractions that
contain metallothionein in normal mice, any l09Cd binding activity associated with KO
mice cytosol should be considered background. These levels do not increase when zinc
treatment is given (data not shown). To better define the metallothionein content of
tissues we deducted the average 109Cd binding value associated with the cytosol of KO
mice from all groups when measuring metallothionein in the intestinal mucosa (0. 1 mg/g
protein; 15 nmol/g protein) and liver (10 ug/g liver; 1.5 nmol/g liver). It is clear that the
saline treated controls have little metallothionein protein present in intestinal mucosa
(Fig. 2-3). TG mice, however, have significantly elevated metallothionein levels
compared to CT mice. Zinc treatment significantly increased metallothionein in all but

1.2

c 1°

] Saline
1 Zinc

o 0.8

o) 0.6

I—

5 0.4
E 0.2

* i

0.0

r— n ■

■ ~*~ '

] Saline
I Zinc

Figure 2-3. Metallothionein content of intestine mucosa of metallothionein-transgenic
and metallothionein-knockout mice after an oral zinc dose. Metallothionein (MT) was
measured by the l09Cd binding assay (mg MT/g mucosa protein) in the intestine mucosa
of metallothionein-transgenic (TG), TG control (CT), metallothionein-knockout (CK),
and KO control (CK) mice after an oral dose of either saline (□) or saline and 0.5 mmol
Zn/kg bw (■). Data are reported as mean +/- SEM of 4-5 mice/group. A constant value
of 0.1 mg MT/g mucosa protein was deducted from all measurements to account for the
nonspecific 109Cd binding observed in KO mouse mucosa. Data was analyzed by
ANOVA followed by the Student-Newman-Keuls multiple comparison post test. Bars
labeled with different letters within a graph are statistically different from each other (p
< 0.05)

the KO mice group. Again, the induction was greatest in TG mice, being eight fold
higher than the zinc treated CT mice. Overall metallothionein protein was not present in
KO mice, was only present in CT and CK mice after zinc treatment, and was always
greatest in TG mice.

Metallothionein was also measured in liver (Fig. 2-4). Unlike in the intestine,
liver metallothionein in saline treated controls was greater than in KO mice, and similar
to TG mice. Zinc treatment elevated the mean value of metallothionein 5-fold in CT (p >
0.05) and 10-fold in TG mice livers, but the increase was only significant in TG mice. No
increase in liver metallothionein was seen in CK mice after zinc treatment. This was not
completely unexpected, however, since differences in zinc induction of liver

180
150

~ 1
CO

■i 90"
60
30
0

] Saline
I Zinc

CKO

CTG

Figure 2-4. Metallothionein content of liver in metallothionein-transgenic and
metallothionein-knockout mice after an oral zinc dose. Metallothionein (MT) was
measured by the i09Cd binding assay (ug MT/g liver) in the liver of metallothionein-
transgenic (TG), TG control (CT), metallothionein-knockout (KO), and KO control
(CK) mice 2 h after an oral dose of either saline (□) or saline and 0.5 mmol Zn/kg bw
(■). Data are reported as mean +/- SEM of 4-5 mice/group. A value of 10 ug MT/g
liver was deducted from all measurements to account for the nonspecific 109Cd binding
observed in KO mouse liver. TG and CT data was logl0 transformed prior to statistical
analysis to achieve homogeneous variances. Data was analyzed by ANOVA followed
by the Student-Newman-Keuls multiple comparison post test. Bars labeled with
different letters within a graph are statistically different from each other (p < 0.05).

metallothionein have been seen among mouse strains (Fair and Hunt 1989). Hence, each
group had a different amount of metallothionein protein present in intestine and liver
after zinc treatment. Since metallothionein may provide a zinc storage pool in these
organs, mice with the greatest metallothionein production (TG mice) have a greater
capacity to deal with a zinc load than those with the least metallothionein (KO mice).

Intestine, Liver and Serum Zinc Responses Two Hours after the Oral Zinc Dose

Although metallothionein in intestine and liver differed among groups, these
differences were not correlated to detectable differences in intestine and liver zinc
concentrations (data not shown). When saline was given, all mice had roughly equivalent
zinc concentrations in both intestine and liver (approximately 0.48-0.62 umol Zn/g and
0.37-0.59 umol Zn/g, respectively). No significant elevation of liver zinc was seen after
zinc treatment. All groups had significantly elevated zinc concentrations in intestine after
zinc treatment, but there were no significant differences among the zinc-treated groups
except in the KO group. In the KO mice, zinc treatment increased the intestinal zinc
concentration significantly compared to the zinc-treated CK mice (1.37 ± 0.22 vs. 0.86 ±
0. 1 1 umol Zn/g, respectively; p < 0.05). In contrast, the intestinal zinc concentrations in
zinc-treated CT and TG mice were similar (0.97 ±0.18 and 1.03 ± 0.18 umol Zn/g,
respectively). Hence, absence of metallothionein resulted in a detectable increase in zinc
accumulation in intestine. However, overexpression in the TG mice did not influence
intestinal zinc retention. This suggests that metallothionein does not alter zinc
metabolism simply by sequestering zinc in the intestine. The change in serum zinc
concentration two hours after the oral zinc dose was used as an indicator of the quantitiy
of zinc absorbed (Fig. 2-5). In contrast to tissue zinc, serum zinc was markedly affected

by metallothionein expression. Although all groups had similar serum zinc
concentrations when given saline (15-30 umol/L), mice with greater metallothionein
expression had lower concentrations after zinc treatment than mice with less
metallothionein expression. Zinc treated control strains had serum zinc concentrations 4
to 5 times higher than saline treated controls. KO mice, however, had 10-fold greater
serum zinc values after zinc treatment. Conversely, TG mice had only 2.3-fold greater
serum zinc concentrations after zinc treatment. Thus, serum zinc concentrations were
inversely proportional to the amount of intestinal metallothionein expressed. This
relationship is illustrated in Fig. 2-6.

Discussion
Our hypothesis was that intestinal metallothionein acts as a negative regulator of
zinc absorption. This relationship has been examined in the past, but with conflicting

Figure 2-5. Serum zinc in metallothionein-transgenic and metallothionein-knockout
mice after an oral zinc dose. Zinc was measured by atomic absorption
spectrophotometry (umol zinc/L) in serum of metallothionein-transgenic (TG), TG
control (CT), metallothionein-knockout (KO), and KO control (CK) mice 2 h after and
oral dose of either saline (□) or saline and 0.5 mmol Zn/kg bw (■). Data are reported
as mean +/- SEM of 4 mice/group. Data were analyzed by ANOVA followed by the
Student-Newman-Keuls multiple comparison post test. Bars labeled with different
letters within a graph are statistically different from each other (p < 0.05).

results. For instance, Dr. Cousins' laboratory (Hoadley et al. 1987, Menard et al. 1981,
Smith and Cousins 1980, Smith et al. 1978) found that the quantity of zinc absorbed by
the isolated perfused rat intestine was inversely related to the zinc content of the diet
consumed prior to the experiments. In addition, giving rats a large zinc dose (i.p.) 18 h
prior to experiments depressed zinc absorption. Since zinc absorption was inversely
proportional to intestinal metallothionein throughout those experiments, it was proposed
that metallothionein serves as a damper of zinc absorption. Similarly, studies where rats
consumed diets ranging from 5 to 80 mg Zn/kg also showed that zinc absorption was
inversely related to metallothionein-bound zinc (Coppen and Davies 1987). Furthermore,
Hoadley et al. (1988) found that elevated metallothionein levels in intestines of fasted
rats were associated with greater mucosa to lumen transfer of absorbed zinc by the
isolated perfused rat intestine. They proposed that metallothionein depresses zinc
absorption by providing a sink that holds zinc in the intestine, allowing more opportunity
for transfer of zinc back into the lumen.

Contrary to the results cited above, other studies found no relationship or positive
correlation between intestinal metallothionein and zinc absorption. For example, bacterial
infection, endotoxemia, and interleukin- 1 administration to rats all elevated liver
metallothionein, and resulted in 50-100% greater zinc absorption and liver zinc
accumulation from 65Zn doses (Kincaid et al. 1976, Pekarek and Evans 1975, 1976).
Intestinal metallothionein expression was not evaluated in those experiments, but
endotoxin has been shown to induce both metallothionein- 1 and metallothionein-2 in
mouse intestine (De et al. 1990). Also, interleukin- 1 is thought to be a mediator of
metallothionein induction by LPS in some tissues, and thus may induce the protein in the

intestine as well (reviewed in Cousins 1996). Furthermore, small zinc doses (0.2 umol/kg
body weight; i.p.) caused metallothionein induction in the mouse intestine and
corresponded to enhanced zinc absorption 18 h later (Starcher et al. 1980). Flanagan and
coworkers (1983) observed no difference in zinc uptake or absorption in relation to
intestinal metallothionein in intestinal perfusion experiments with mice. They did,
however, see greater zinc absorption in zinc-deficient vs. control mice when doses of

X KO

• CK
A CT

♦ TG

Intestine Metallothionein (mg/g protein)

Figure 2-6. Serum zinc concentration as a function of intestine metallothionein content
in zinc-treated mice. Response of serum zinc (Y) vs. intestinal metallothionein content
(X) in metallothionein-transgenic (TG), TG control (CT), metallothionein-knockout
(KO), and KO control (CK) mice 2 h after gavage with 0.5 mmol Zn/kg bw.
Metallothionein (MT) is expressed as mg MT/g mucosa protein and serum zinc as
umol Zn/L. The inverse relationship between intestinal MT and serum zinc can be
expressed mathematically with good fit (Y = 1690/X + 0.75; r2 = 0.94).

zinc were delivered by gavage. They also demonstrated that differences exist in zinc
absorption characteristics between rats and mice, particularly the response of increased
absorption during zinc deficiency.

Although the studies cited above focused on the effect of metallothionein on zinc
absorption, the methods used to alter intestinal metallothionein levels varied. Treatments
used to induce the protein included intraperitoneal, intragastric, and dietary doses of zinc,
fasting, bacterial infection, lipopolysaccharide and interleukin-1 administration, and
various forms of physical stress. Although these treatments manipulate metallothionein
expression, each has effects not related to this protein that may cause physiological
changes and complicate interpretation of the results. Using knockout and transgenic
mouse models, it is possible to focus on zinc absorption as directly related to
metallothionein expression.

Giving animals a large oral dose of zinc by gavage, we were able to determine the
effects of metallothionein induction on zinc absorption by measuring serum and tissue
zinc concentrations. This avoids the potential for isotope dilution, which can cloud
interpretation of radioisotopic tracer studies using 65Zn. We have used the oral dosing
approach previously (Menard et al. 1981). It is equivalent to the zinc tolerance test used
with humans (Sullivan et al. 1979). We used fasting and dosing in saline to prevent
nonspecific binding of zinc to food in the gut, and to allow for a maximal gastric
emptying rate. The 2 h time point used was determined to be the time point of maximal
serum zinc response in these mouse strains (data not shown), and agrees with data from
rats (Menard et al. 1981) and humans (Sullivan et al. 1979, Valberg et al. 1985). Further,
all dosing was done between 8 AM and 10 AM. The 0.5 mmol/kg dose given is 2.5 to 3.1

times greater than the typical dietary zinc intake of these mice (0.17 to 0.22 mmol/kg
body weight). Although greater than the typical intake, this dose is attainable through the
diet, and is therefore nutritionally relevant. Further, since the dose produced fivefold to
tenfold increases in serum zinc, we anticipate that smaller doses will also result in
significant differences, albeit smaller. Menard et al. (1981) showed that intestinal
metallothionein synthesis in rats was increased by 3 h after an oral zinc dose is given.
Furthermore, a second dose of zinc caused induction of metallothionein synthesis and
resulted in better regulation of serum zinc concentrations. In the present experiments,
serum zinc doubled in TG mice and increased 10-fold in KO mice when zinc was
delivered by gavage. We interpret the inability of the KO mice to handle the zinc load
compared to the TG mice to the difference in metallothionein expression. Specifically,
the TG mice controlled serum zinc concentrations more tightly than did the KO mice.
Serum zinc concentrations remain elevated for a considerable time after the oral dose.
We cannot rule out that the observed differences wee related to different kinetics of
absorption in these genotypes, however.

A drawback of this approach is that the role of other tissues in clearance of zinc
from the circulation cannot be explained adequately. Since the gene addition in TG mice
and gene deletion in KO mice are not tissue specific, we cannot rule out the possibility
that MT expression in some other tissue affected zinc clearance from the serum.
However, we did measure the zinc content of the liver, the main zinc storage organ and
the key organ in the regulation of zinc metabolism (Cousins 1996, Coyle et al. 1995). In
this study, no change in liver zinc was detected between saline or zinc-treated mice, and
no difference was seen among groups of zinc-treated mice. This is in agreement with data

collected in rats, where hepatic accumulation of gavaged zinc was not observed until nine
hours after dosing (McCormick et al. 1981). Since the liver can act rapidly to regulate
zinc metabolism, yet did not show an elevation in zinc concentration, it is unlikely that an
organ with less influence on zinc metabolism caused the differences seen in serum zinc.

In the time that has elapsed since the publication of the results found above
(Davis et al. 1998), several reports have been forwarded regarding the role of
metallothionein in zinc absorption. A number of these studies used a separate strain of
metallothionein knockout mice (Michalska and Choo 1993). Our findings regarding the
serum zinc response to the oral zinc dose was confirmed in these studies, i.e., knockout
mice had greater serum zinc levels than control mice over a range of oral doses that
began two orders of magnitude lower than in our studies (Coyle et al. 1999). Further,
their studies confirmed greater zinc accumulation in duodenum and jejunum of KO mice
than CK mice. These researchers also found lower accumulation of a radiolabelled dose
in tissues other than the intestine (liver, skin, muscle, kidneys and pancreas) at 4 h post
dose, which they interpreted as reduced absorption. However, KO mice lack the ability to
sequester zinc in several organs (as determined by the researchers mentioned above, as
well as by us in chapters 3, 4, and 5 of this dissertation), including the liver, which
renders measurement of tissue zinc accumulation unsuitable as an index of intestinal zinc
absorption in this model.

Within intestinal cells, higher intakes of zinc may be processed via a mechanism
that involves metallothionein. Since elevated intestinal metallothionein levels were not
associated with greater intestinal zinc accumulation, metallothionein does not seem to act
simply as a zinc sequestrant. Metallothionein may act as a zinc pool from which zinc is

highly available for transport back to the lumen, as suggested by Hoadley et al. (1988).
Without metallothionein, the KO mice may be unable to maintain a satisfactory
mucosa-to-lumen zinc flux. This might explain why KO mice have elevated zinc levels in
serum and intestine. Since metallothionein mRNA levels are also induced by zinc in
humans (Sullivan and Cousins 1997), we expect that metallothionein would affect zinc
absorption by the human intestine as well.

CHAPTER 3

REGULATION OF METALLOTHIONEIN EXPRESSION AND ZINC METABOLISM

BY DIETARY ZINC IN METALLOTHIONEIN TRANSGENIC AND

METALLOTHIONErN KNOCKOUT MICE

Introduction

Homeostatic regulation of zinc metabolism by the dietary zinc supply is believed
to involve the protein metallothionein (as reviewed in Davis and Cousins 2000).
Metallothioneins are cysteine-rich, low molecular weight (6-7 kDa) metal binding
proteins that can bind up to seven atoms of zinc per molecule of protein (as reviewed in
Dunn et al. 1987). Metallothioneins are thought to provide a cellular zinc binding pool
that may be influenced by body zinc levels (as reviewed in Davis and Cousins 2000).
Redistribution of endogenous zinc is associated with stresses such as acute infection and
physical trauma. Redistribution is believed to involve IL-1, IL-6, and glucocorticoid
hormone mediated changes in zinc metabolism, all of which are linked to elevated
expression of metallothionein in the liver and other tissues (reviewed in Cousins 1989
and 1996). Metallothionein may also be involved in regulation of dietary zinc absorption
in the intestine, and affect accumulation of dietary zinc in the liver (reviewed in Davis
and Cousins 2000). Specifically, induction of intestinal metallothionein may reduce the
efficiency of zinc absorption during times of elevated zinc intake.

Previous studies firmly established the association of metallothionein induction
with cellular zinc accumulation and bodily zinc redistribution (reviewed in Davis and
Cousins 2000). Recent studies using metallothionein overexpressing transgenic mice (TG

mice) and metallothioein knockout mice (KO mice) have more directly analyzed the role
of metallothionein in zinc metabolism (Masters et al. 1994, Palmiter et al. 1993). Results
from these studies confirmed the role of metallothionein expression in protection against
severe zinc deficiency (Andrews and Geiser 1999, Dalton et al. 1996, Kelly et al. 1996).
Other studies confirmed the necessity of metallothionein expression for zinc
redistribution in response to immune stress, oxidative stresses and fasting (Davis et al.
submitted, Philcox et al. 2000, Rofe et al. 1996). Metallothionein 's relationship to
suppression of zinc absorption is still under debate (Davis et al. 1998, Coyle et al. 1999).
Questions remain regarding metallothionein's role in tissue distribution of dietary
zinc. Previous studies from this lab found that metallothionein induction after acute
elevations in zinc intake was associated with accumulation of zinc in rat liver and
intestine. Whether metallothionein induction is responsible for this increase in hepatic
zinc, or whether it is only responding to the elevated hepatic zinc in not clear. Further,
the effect of metallothionein expression on zinc distribution in mice during chronic
exposure to elevated dietary zinc intakes is unknown. In these experiments we studied the
interrelationship of metallothionein expression and chronic exposure to a spectrum of
dietary zinc intake levels in metallothionein transgenic and metallothionein knockout
mice in order to monitor this mode of regulation more closely. The results suggest that
metallothionein expression affects tissue zinc accumulation only at highly elevated zinc
intakes. Metallothionein appeared to regulate its own expression, however. This might
have occurred through greater accumulation of cytosolic zinc. If so, metallothionein
expression may alter the expression of other zinc-regulated genes.

Materials and Methods
Animals

Metallothionein knockout and metallothionein transgenic mice used in this study
were derived from founder mice purchased from The Jackson Laboratory (Bar Harbor,
ME). The metallothionein overexpressing mice (designated TG mice) were generated in
C57BL/6 mice crossed with SJL mice (Palmiter et al. 1993), whereas metallothionein
knockout mice (designated KO) were generated in 129/SvCPJ mice (Masters et al. 1994).
C57BL/6 mice (designated CT) and 129S3/SvCPJ mice (designated CK) served as
controls, respectively. Only 7-9 week old male mice were used in these experiments.

Experimental Design

Mice were housed singly in stainless steel hanging cages with a 12 h lightdark
cycle. During experiments mice were given access to deionized water and semipurified
diet based on the AIN-76A formulation (AIN 77; Research Diets, New Brunswick, NJ)
with one of five zinc contents: 10, 50, 100, 200 and 300 mg zinc/kg diet (designated Zn10,
Zn50, Zn100, Zn200 and Zn300, respectively). Initially, mice were given free access to the
Zn10 diet and deionized water for seven days. For seven days thereafter mice were given
free access to the Zn10, Zn50, Znl00, Zn200, or Zn300 diet. All mice were killed between 8
AM and noon following completion of the dietary treatment. Care and treatment of the
mice received approval of the University of Florida Institutional Care and Use
Committee.
RNA Isolation and Northern Analysis

Total RNA was isolated from intestine and liver using TRIzol reagent (Life
Technologies, Gaithersburg, MD). Briefly, 50-100 mg of the proximal duodenum and

the liver were homogenized in 2 mL of TRIzol reagent. After addition of chloroform, the
RNA was processed and analyzed as described previously (Davis et al. 1998). Equal
quantities of RNA from mice of each group were pooled and subjected to northern
analysis (20 ug total RNA per lane). Equal loading was confirmed by ethidium bromide
staining. Northern blot analyses were carried out using a rat metallothionein-1 cDNA
probe (Blanchard and Cousins 1996). This was radiolabeled with a32P-dCTP (Du Pont
NEN, Boston, MA) using the RTS RadPrime DNA Labeling System (Life Technologies)
as described previously (Blanchard and Cousins 1996). The metallothionein cDNA probe
hybridizes to both the normal metallothionein mRNA of the control and TG mice, and
the disrupted MT mRNA of the KO mice. Northern blots were also hybridized with a (3-
actin probe, and the P-actin signal was used for normalization. Densitometry of the
autoradiographs was performed by scanning the film and measuring the relative intensity
using Intelligent Quantifier software (Bio Image, Ann Arbor, MI).

Analytical Methods and Statistical Analysis

Metallothionein was measured as described previously (Davis et al. 1998) using
the cadmium (109Cd) binding assay (Eaton and Toal 1982). Total protein was measured
by the method of Lowry ( 1 95 1 ). Serum zinc concentrations were measured by flame
atomic absorption spectrophotometry (AAS) (Hempe and Cousins 1989) after dilution
with deionized water. Tissue zinc was measured by AAS after sections of liver and
intestine were digested with HN03/H2S04 (3/1) as described previously (Dunn and
Cousins 1989). Data were analyzed by two way ANOVA (2x5) with SigmaStat software
(Jandel Scientific, San Rafael, CA) to determine specific main effects and interactions
using genotype and dietary zinc as independent variables using. The Tukey-Kramer

multiple comparison test was used to determine significant differences between specific
groups (p < 0.05).

mg Zn/kg diet
MT- 1 mRNA

(3-Actin mRNA

mg Zn/kg diet
MT-1 mRNA

(3-Actin mRNA

10 50 100 200 300

0 50 100 200 300

10 50 100 200 300

10 50 100 200 300
mg zinc/kg diet

Figure 3-1. Intestinal metallothionein mRNA in metallothionein knockout (KO),
knockout control (CK), transgenic (TG), and transgenic control (CT) mice that consumed
diets containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. Equal amounts of total
RNA were pooled from intestine of 3-5 mice per group and analyzed by northern analysis
using a metallothionein- 1 cDNA probe, with (3-Actin used for normalization. (A) KO and
CK mouse intestine. Although metallothionein- 1 mRNA is present in KO mice, it is not
translatable and therefore does not give rise to metallothionein protein. (B) CT and TG
mouse intestine. (C) Graphical representation of metallothionein- 1 mRNA expression as
a function of dietary zinc content.

Results

Intestinal metallothionein mRNA was visibly upregulated by dietary zinc in CK
and CT mice fed the Zn100 through Zn300 diets (Fig. 3-lA,B). Dietary zinc induced
metallothionein mRNA more strongly and at lower zinc intakes in TG mice, but had little
effect on metallothionein mRNA in KO mice. The proportional increases in
metallothionein mRNA expression with dietary zinc intake were greater in mice with
greater metallothionein expression (TG > CT and CK > KO) (Fig. 3-1C). The
metallothionein mRNA expression pattern was similar in liver of KO, CK and CT mice
(Fig. 3-2A,B). Metallothionein mRNA expression in TG liver was very high at all dietary
zinc intakes, but relatively constant until the Zn300 diet was consumed.

Intestinal metallothionein protein was measured in all genotypes (Fig. 3-3A,B).
The minimal values for metallothionein protein in KO mice (Fig. 3 -3 A) were shown to
be assay background only (Davis et al. 1998). Considering this, little metallothionein
expression was detected in CT or CK mice consuming the Zn10, Zn50 or Zni00 diets (Fig.
3-3 A,B). Expression of metallothionein protein was directly regulated by dietary zinc in
mice consuming the Zn200 and Zn300 diets. Similar to the response of metallothionein
mRNA, intestinal metallothionein protein was induced at lower dietary zinc intakes and
to a greater extent in TG mice.

Intestine zinc concentrations were significantly affected by dietary zinc (p =
0.00008) in CT and TG mice. Intestine zinc contents appear to be associated with
intestinal metallothionein protein levels (Fig. 3-3A-D). Zinc content started to increase in
all mouse intestines at the same dietary zinc level that metallothionein induction

occurred. The increase in intestine zinc was greater in TG mice (except with the Zn300
diet), and no increase was seen in KO mice.

In contrast to the above results, the effects of dietary zinc on metallothionein
expression and zinc content in liver differed between the two control species (Fig. 3-4A-
D). Dietary zinc did not affect liver metallothionein levels in CK or KO livers (Fig. 3-
4A). Also, metallothionein levels in CK mice did not exceed the assay background seen
in KO mice. In contrast, TG mice had greater liver metallothionein values than CT mice
at all zinc intakes, but zinc did not induce metallothionein above basal levels until diets
containing 300 mg Zn/kg were consumed (Fig. 3-4B).

Liver zinc was not altered by dietary zinc or metallothionein expression except at
the highest dietary zinc intake (Fig. 3-4C,D). Liver zinc was not affected by dietary zinc
or genotype in CK and KO mice (Fig. 3-4C). Only the high dietary zinc concentration
(Zn300) elevated liver zinc, and only in TG mice (Fig. 3-4D).

mg Zn/kg diet

MT-1 mRNA

P-Actin mRNA

mg Zn/kg diet
MT- 1 mRNA

(3-Actin mRNA

CK KO

10 50 100 200 300 10 50 100 200 300

CT TG

10 50 100 200 300 10 50 100 200 300

« liM|

Figure 3-2. Liver metallothionein mRNA in metallothionein knockout (KO), knockout
control (CK), transgenic (TG), and transgenic control (CT) mice that consumed diets
containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. mRNA was analyzed by
northern analysis as described in Fig. 3-1. (A) KO and CK mouse intestine. (B) CT
and TG mouse liver.

Serum zinc was affected by dietary zinc in all genotypes (Fig. 3-5 A,B). Serum
zinc began to rise in CT and CK mice consuming Zn100 and Zn200 diets, respectively, and

1000

a 800

P. 600

S 400

H 200

I CK

D ko

a a
10

' Dct B

■ TG

a,b
a

aa aj ftj

c
1

300

10 50 100 200 300

3000
2500

M 2000

2 1500

3 1000

500

I CK

□ KO

tad

10 50 100 200 300 10 50

mg zinc/kg diet

100 200 300

Figure 3-3. Intestinal mucosal metallothionein protein and zinc in metallothionein
knockout (KO), knockout control (CK), transgenic (TG), and transgenic control (CT)
mice that consumed diets containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d.
Mucosal metallothionein in (A) CK and KO mouse intestine or (B) CT and TG mouse
intestine. Values found for KO mice reflect assay background only. Intestinal zinc in
(C) CK and KO mouse intestine or (D) CT and TG mouse intestine. Data are
presented as means ± SE of 3-6 mice/group. Statistical differences (p < 0.05) were
determined by two-way ANOVA followed by the Tukey-Kramer post test

continued to rise with increasing dietary zinc level. The effect of diet alone was very
significant (p = 0.0000005). No differences were seen between CK and KO mice. Serum
zinc of TG mice was slightly elevated comparedto CT mice consuming Zn10-Zn200 diets.
When Zn300 diets were consumed, however, serum zinc was greater in CT mice than TG
mice.

Discussion
Metallothionein has long been implicated as a key biomolecule in regulation of
zinc homeostasis. Specifically, many lines of evidence point to metallothionein as a
component of the machinery involved in intracellular zinc accumulation (as reviewed in
Davis and Cousins, 2000). Here, we confirm that KO mice are unable to accumulate zinc
in the intestine in response to chronic exposure to elevated dietary zinc, as was found
previously in a different metallothionein KO mouse model (Tran et al. 1998). This report
extends this relationship to TG mice, which generally accumulated more intestinal zinc
than controls. These data show that zinc accumulation is affected by metallothionein
expression over a wide range of intestinal metallothionein contents. Further, our data
from TG mice show for the first time that the level of hepatic metallothionein expression
dictates the livers ability to accumulate zinc during chronic exposure to elevated dietary
zinc. This expands on the relationship previously seen after zinc injections in control and
KO mice, where KO mice were unable to accumulate zinc in the liver (Rofe et al. 1996).
If hepatic uptake of zinc is important during infection and trauma, metallothionein might
be crucial for processes that depend on this influx of zinc. Such a role has been suggested
for metallothionein expression in rat liver during regeneration and the acute phase
response (Arora et al. 1998, Dunn and Cousins 1989, Ohtake et al. 1978). A similar role

would be predicted in other metallothionein-expressing organs. As such, overexpression
of metallothionein may be beneficial to liver regeneration, or conditions associated with
the acute phase response.

We also discovered that induction of liver and intestine metallothionein mRNA
and protein occurred at lower dietary zinc intakes in animals with greater metallothionein

200

10 50 100 200 300

2000
1800
1600
1400

g 1200

jj 1000-
o

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c

800
600
400
200

■ CK
□ KO

miiil

10 50 100 200 300 10 50

mg zinc/kg diet

100

200 300

Figure 3-4. Liver metallothionein protein and zinc in metallothionein knockout (KO),
knockout control (CK), transgenic (TG), and transgenic control (CT) mice that consumed
diets containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. Metallothionein in (A)
CK and KO mouse liver or (B) CT and TG mouse liver. Values found for KO mice
reflect assay background only. Zinc concentration in (C) CK and KO mouse liver or (D)
CT and TG mouse liver. Data are presented as means ± SE of 3-6 mice/group. Statistical
differences (p < 0.05) were determined by two-way ANOVA followed by the Tukey-
Kramer post test.

production (TG < CT and CK < KO; Fig. 3-1 & Fig. 3-2). Similar results were seen for
metallothionein protein (Fig. 3-3A,B & 3-4A,B). These data suggest that metallothionein
enhances its own expression, possibly by facilitating cellular zinc accumulation. In this
hypothesis, accumulation of cellular zinc in the metallothionein-zinc pool provides a
labile zinc pool that may interact with other pools (as illustrated in Davis and Cousins
2000). One such pool is the nucleus, where dietary zinc rapidly accumulates in a number
of organs (Cousins and Lee- Ambrose 1992). One of the biomolecules that the
metallothionein-Zn pool may interact with is the zinc-finger transcription factor MTF-1.
MTF- 1 contains a zinc-binding site that may be sensitive to cellular zinc levels (as
reviewed in Andrews 2000). Zinc from the metallothionein-Zn pool may activate MTF-1,
resulting in subsequent transcriptional activation of the metallothionein gene through
numerous metal response elements (MREs) in the metallothionein gene promoter. This

10 50 100 200 300 10 50 100 200 300

mg zinc/kg diet

Figure 3-5. Serum zinc in metallothionein knockout (KO), knockout control (CK),
transgenic (TG), and transgenic control (CT) mice that consumed diets containing
either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. Serum zinc in (A) CK and KO mice
or (B) CT and TG mice. Data are presented as means ± SE of 4-6 mice/group.
Statistical differences (p < 0.05) were determined by two-way ANOVA followed by
the Tukey-Kramer post test.

process would enhance production of metallothionein protein, which, after further zinc
binding, may fuel even greater metallothionein gene transcription. Cycling through this
pathway would amplify metallothionein levels, and as we observed, more amplification
would occur in mice with greater metallothionein expression.

The fact that metallothionein protein expression was elevated only with chronic
dietary zinc supplementation is in line with previous mouse experiments (Olafson 1983,
Tran et al. 1998). Tissue zinc levels were only elevated at high zinc intakes, also. Further,
KO mice maintained control over serum zinc, liver zinc and intestine zinc levels as well
as CK mice did. These results suggest that mice can maintain tight control of zinc
homeostasis over a large range of zinc intakes without metallothionein induction. Many
zinc transporter molecules have been identified recently, and it is likely that regulation of
the activity or expression of those proteins mediated zinc homeostasis over that range of
zinc intakes (reviewed in McMahon and Cousins 1998, Cousins and McMahon 2000).
The high correlation of zinc transporter 1 expression (Davis et al. 1998) and zinc
transporter-2 expression (Liuzzi et al. 2000) with metallothionein expression supports the
involvement of zinc transporters and metallothionein expression in zinc homeostasis.
Metallothionein appears to become an important mediator of cellular zinc homeostasis at
very high dietary zinc intakes, however (Davis et al. 1998). This is not to say that
metallothionein plays no role in zinc homeostasis at lower dietary zinc levels. It has been
shown that metallothionein expression provides partial protection against zinc deficiency
(Andrews and Geiser 1999, Dalton et al 1996, Kelly et al. 1996). It also is necessary for
redistribution of zinc during periods of stress (Cousins and Leinart 1988, Davis et al.
submitted, Dunn and Cousins 1989, Philcox et al. 1995). When considering the typical

range of dietary zinc intakes and in the absence of metallothionein-inducing stresses,
however, the effect of metallothionein expression on zinc homeostasis is minimal.

In conclusion, we found that metallothionein expression alters tissue zinc
accumulation, but only at highly supplemental zinc intakes. At lower intakes
metallothionein was not a factor. We found evidence that metallothionein expression
might act in a positive feedback loop to regulate its own expression, and that this occurrs
to a greater degree in mice with a greater number of MT gene copies. If this hypothesis is
true, metallothionein may also be involved in regulation of other zinc responsive genes.

CHAPTER 4
THE EFFECTS OF METALLOTHIONEIN GENE EXPRESSION AND

SUPPLEMENTAL DIETARY ZINC IN PROTECTION AGAINST

HEPATOTOXICITY IN METALLOTHIONEIN TRANSGENIC AND

METALLOTHIONEIN KNOCKOUT MICE

Introduction

Zinc and metallothionein are implicated in cellular defense against a number of
cytotoxic agents. There is evidence that supplemental zinc and overexpression of
metallothionein help protect cells and organisms from a number of stresses. For example,
administration of pharmacological zinc doses protects rodents from the toxicity of certain
metals and other chemicals, some of which cause oxidative stress (Blain et al. 1998,
Chvapil et al. 1973, Dhawan and Goel 1995, Powell et al. 1994). Similar results were
seen in cell culture (Coppen et al. 1988, Liu et al. 1991, Tate et al. 1999). The
mechanism(s) through which zinc provides protection is uncertain. Zinc may protect
sulfhydryl groups from oxidation, may limit the redox reactive metal content of tissues,
or may elevate the activity of antioxidant enzymes (Coppen et al. 1988, Davis, C. D. et
al. 2000, Olin et al. 1995). Many believe that supplemental zinc provides antioxidant
protection through its powerful induction of metallothionein gene expression.

Preinduction of metallothionein by a number of metals (including zinc),
hormones, cytokines and other chemicals is associated with protection from the toxicity
of subsequent metal, chemical, and other stresses in cell culture and in vivo (Blain et al.
1998, Coppen et al. 1988, Kelley et al. 1988, Liu et al. 1991, Mello-Filho et al. 1988,
Moffat et al. 1996, Naganuma et al. 1985, Satoh et al. 1988, Schroeder and Cousins

1990). Several experiments in cell cultures transfected with metallothionein genes (Kaina
et al. 1990, Schwarz et al. 1995, Yao et al. 2000) and in cultures from metallothionein
transgenic and knockout mice (Lazo et al. 1995, Kondo et al. 1995, Wang et al. 1999,
Zheng et al. 1996) found similar results. Finally, a number of experiments in
metallothionein transgenic and metallothionein knockout mice came to similar
conclusion (Kang et al. 1997, Kang et al. 1999, Liu et al. 1995, Liu et al. 1998A, Liu et
al. 1999A, Masters et al. 1994, Michalska and Choo 1993, Rofe et al. 1998). The results
of several papers contradict the idea that metallothionein is universally protective,
however (DiSilvestro et al. 1996, Itoh et al. 1997, Liu et al. 1999A, Minami et al. 1999).

In some studies, metallothionein induction by zinc protected against the free
radical generating hepatotoxin carbon tetrachloride. These conclusions were reached
from in vitro studies, in vivo studies using nonspecific inducers of metallothionein, and
studies using pharmacological injections of zinc. While injections of zinc are known to
induce metallothionein and protect against oxidative stress in liver and cultured cells, it is
not clear whether supplemental dietary zinc mimics the protective effects of parenterally
administered zinc, and if so, whether the protection depends upon metallothionein
production. Conversely, it is not known whether the effect of metallothionein induction
on cytoprotection depends on the level of dietary zinc. Further, whether supplemental
zinc and metallothionein expression act additively or synergistically is uncertain.

Murine metallothionein knockout and metallothionein overexpressing models are
more direct models of the effects of metallothionein expression on cytotoxicity. Also,
zinc presented via the diet is more physiological than via injection. We determined
whether metallothionein overexpressing mice or metallothionein knockout mice had

altered sensitivity to carbon tetrachloride compared to identically treated controls. We
also determined whether supplemental dietary zinc reduced sensitivity to carbon
tetrachloride in these genotypes.

The results of the experiments explained herein confirm the importance of
metallothionein expression in protection against oxidative stress, but bring into question
the impact of supplemental zinc and/or elevated metallothionein expression in defense
against oxidative stress in vivo.

Materials and Methods

Animals

Metallothionein overexpressing mice (TG mice), C57BL/6 mice (CT mice),
metallothionein knockout mice (KO mice) and 129/SvCPJ mice (CK mice) were used in
these experiments. All experimental groups were age (8-1 1 wk) and sex matched. Mice
were housed singly in stainless steel hanging cages with a 12 h light:dark cycle. During
experiments, mice were given free access to deionized water and AIN-76A diets with
adequate or supplemental zinc content (10 or 500 mg zinc/kg diet, respectively; Research
Diets, New Brunswick, NJ). Care and treatment of the mice received approval of the
University of Florida Institutional Care and Use Committee.

Experimental Design

TG, KO and control mice were acclimated to diet containing 10 mg zinc/kg (Zn10)
and deionized water for 7 d. Following this acclimation period, one half of the mice were
switched to a diet containing 500 mg zinc/kg (Zn500) for 3 d while the other half remained
on the Zn10 diet. After the third day of dietary treatment, mice were injected with carbon

tetrachloride (CC14) in corn oil (20 ul/kg bw, i.p.) or corn oil alone between 8 AM and 10
AM, and animals were killed at 0 h, 12 h, 24 h, and 48 h post dose. Since mice in the 0 h
group did not receive injections, data at this time point represent the effects of diet and
genotype only.

Food Intake and Body Weight

Food intake was measured for the 3 d dietary treatment. Food intake intake is
expressed as mg/g body weight. Body weight was measured at the onset of the 3 d
dietary treatment.

Analytical Methods

109Cd was measured using a Packard Cobra II gamma spectrometer equipped with
a 3-inch crystal (Packard, Downers Grove, IL). Metallothionein protein was measured
by the cadmium (109Cd) binding assay (Eaton and Toal 1982). Total protein was
measured by the method of Lowry et al. (1951). Serum zinc concentrations were
measured by flame atomic absorption spectrophotometry (AAS) (Hempe and Cousins
1989). Tissue zinc was measured as described previously (Dunn and Cousins 1989).
Serum alanine aminotranferase (ALT) enzyme activity was measured
spectrophotometrically as the formation of pyruvate from alanine and a-ketoglutarate
using a commercial diagnostic kit (Sigma 505-P). The pyruvate formed is reacted with
2,4-dinitrophyenylhydrazine, forming a 2,4-dinitrophenylhydrazone derivative that can
be measured spectrophotometrically (A.max = 505 nm). The absorbance is converted to
ALT activity units using a standard curve generated for pyruvate.

Total thiol groups were measured in liver homogenates spectrophotometrically
after treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) (Jocelyn 1989).
Reaction of sulfhydryl groups with DTNB generates a yellow chromophore (A.max = 412
nm, extinction coefficient of e ■ 13100 M"1 cm'1). Nonprotein thiols were measured by
the same technique after first removing protein thiols by TCA precipitation (5% TCA).

Histological Analysis of Liver

Sections of liver were fixed in 10% buffered formalin, embedded in paraffin, and
stained with hematoxylin and eosin. These sections were analyzed visually for necrosis
and other signs of hepatotoxicity (Khoo et al. 1996). Micrographs were obtained with a
Zeiss Axiovert SI 00 microscope (Carl Zeiss, Thornwood, NY) fitted with a CCD camera
for processing of digital images.

Statistics

Data were analyzed by ANOVA for a three way factorial design (2x2x2) to
determine significant main effects and interactions using genotype, dietary zinc and
oxidant treatment as independent variables (SAS, SAS Institute Inc. Cary, North
Carolina). The Tukey-Kramer multiple comparison test was used to determine significant
differences between specific groups. Serum ALT data were log transformed to obtain
homogeneous variances. Significance was established at p < 0.05.

A 50

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D 10+CC14
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A 500+CO,

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0 12 24 36 48
Hours

Figure 4-1. Indices of zinc homeostasis in metallothionein knockout (KO) and control
(CK) mice 0, 12, 24, or 48 h after injection of carbon tetrachloride (CC14) or corn oil
(CO). The mice had been fed either adequate dietary zinc (Zn10) or supplemental
dietary zinc (Zn500). (A) Serum zinc concentration (umol Zn/L serum). (B) Liver zinc
(nmol/g liver). (C) Liver metallothionein (nmol/g liver). Data are presented as means
± SE of n = 3 (0 h) or n = 4-7 (12, 24, and 48 h) mice/group. Statistical differences (p
< 0.05) were determined by ANOVA for a three way factorial design, followed by the
Tukey-Kramer multiple comparison test.

Results
Food Intake

Diet consumption was similar between adequate zinc (ZnI0) and supplemental
zinc (Zn500) groups, and was also similar between TG and CT groups (data not shown).
Food consumption was statistically lower in KO than CK mice (p = 0.05), but the
difference was unlikely to have been biologically significant (0.46 mg *g bw"' 3d"1 vs
0.48 mg * g bw"1 3d1).

Zinc Status and Metabolism

Serum zinc, liver zinc and liver metallothionein. were measured at 0 h, 12 h, 24 h,
and 48 h post-dose (Fig. 4-1 & 4-2). Since mice in the 0 h group did not receive
injections, data at this time point represent the effects of diet and genotype only.

Knockout mice. The Zn500 diet significantly increased serum zinc concentration
in both genotypes, but to a greater extent in control (CK) mice (Fig. 4-1 A). The only
significant effect of genotype was seen at 12 h after the CC14 treatment, when the serum
zinc concentration increased by 80% in KO mice only.

At Oh liver zinc content is significantly lower in KO mice than CK mice when fed the
Zn500 diet, but not the Zn10 diet (Fig. 4- IB). Twelve hours later, CK mice fed the Zn500
diet and injected with CO (Zn500+CO) had 25-45% more liver zinc than other CK groups
and significantly more (>100%) liver zinc than all KO groups. This effect may have been
due to the stress of the injections themselves, but all mice received injections at this time
point. The remaining CK groups had 35-90% more liver zinc than KO groups, but the
differences were not statistically significance. At 24 h and 48 h there was significantly
more liver zinc in CK500 mice compared to all other genotype-diet combinations. The

only significant effect of CC14 treatment was at 24 h, when CCl4-treated mice had lower
liver zinc values than CO treated mice. KO mice did not sequester zinc in the liver in

Control

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Figure 4-2. Indices of zinc homeostasis in metallothionein transgenic (TG) and control
(CT) mice 0, 12, 24, or 48 h after injection of carbon tetrachloride (CC14) or corn oil
(CO). The mice had been fed either adequate dietary zinc (ZnI0) or supplemental dietary
zinc (Zn500). (A) Serum zinc concentration (umol Zn/L serum). (B) Liver zinc (nmol/g
liver). (C) Liver metallothionein (nmol/g liver). Data are presented as means ± SE of n =
3 (0 h) or n = 4-7 (12, 24, and 48 h) mice/group. Statistical differences (P < 0.05) were
determined by ANOVA for a three way factorial design, followed by the Tukey-Kramer
multiple comparison test.

response to the Zn500 diet, CC14 treatment, or combination of the treatments.Liver
metallothionein levels depend on genotype and dietary zinc, and also are affected by
oxidant treatment and the stress of the injection (Fig. 4-1C). These values roughly mirror
hepatic zinc values. At 0 h, 150% more metallothionein is detected in CK500 compared to
CK10 mice. At 12 h after injection, CK500+CO mice had greater metallothionein values
than other mice, and CK500 mice had greater values than CK10 mice. Results are similar at
24 h. At 48 h, the metallothionein levels in the CK500+CC14 group increased, and both CK500
groups have five fold greater metallothionein levels than CK,0 groups. As expected, KO
mice did not express metallothionein. It appears that CC14 toxicity delayed the induction
of metallothionein in these mice.

Transgenic overexpressing mice. Zinc homeostasis was also altered by
metallothionein expression. Serum zinc was 80-100% greater in CT mice fed the Zn500
diet than Zn10 diet throughout the experiment (Fig. 4-2A). Serum zinc also rose in TG
mice fed the Zn500 diet, but -50% less than that found in CT mice. At 12 h, there was a
trend toward decreasing serum zinc values after injection of vehicle alone in CT mice,
and both CC14 and vehicle treated TG mice. In contrast, serum zinc values were
significantly greater in CT+CC14 mice at this time point. This is similar to the serum zinc
response of KO+CCL, mice. Throughout the time course of 48 h, TG mice exhibited
better control over the serum zinc concentration, especially when consuming the Zn500
diet. Liver zinc was greater in TG500 mice than CT500 mice at 0 h (Fig. 4-2B). At 12 h,
however, TG500 mice had greater zinc values than all other mice, and TG+CC14 mice had
significantly greater liver zinc than all other genotype-oxidant combinations. At 24 h the

TG500 mice had greater liver zinc values than all other diet/genotype combinations. Liver
zinc declined in all groups between 24 h and 48 h, but remained greatest in TG500 mice.

As with CK and KO mice, liver metallothionein values mirrored the liver zinc
values. TG500 mice had more liver metallothionein at 0 h than other diet-genotype
combinations. Metallothionein was elevated in both genotypes at 12 h after CC14
treatment. Metallothionein was elevated to its highest in all Zn500 groups at 24 h after
injection, and were greatest when the injection contained CC14. At 48 h liver
metallothionein declined in all groups, but the TG500 and TGCC14 groups still had slightly
more metallothionein than all other genotype-diet and genotype-oxidant combinations,
respectively. Interestingly, CC14 alone had a minimal effect on liver metallothionein in
CT or TG mice unless combined with the Zn500 diet.

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Figure 4-3. Serum alanine aminotransferase activity of metallothionein knockout (KO)
and control (CK) mice 0, 12, 24, or 48 h after injection of carbon tetrachloride (CC14) or
corn oil (CO). The mice had been fed either adequate dietary zinc (Zn10) or supplemental
dietary zinc (Zn500). These activities were measured spectrophotometrically and
expressed as log activity units/mL serum. Data are presented as means ± SE of three (0 h)
or 4-7 mice/group (12, 24, and 48 h). Data are presented as means ± SE of n = 3 (0 h) or
n = 4-7 (12, 24, and 48 h) mice/group. Statistical differences (p < 0.05) were determined
by ANOVA for a three way factorial design, followed by the Tukey-Kramer multiple
comparison test.

Hepatotoxicity and Oxidative Stress

Hepatotoxicity was assessed by measuring serum alanine aminotransferase
enzyme (ALT) activity, and by histological analysis of liver sections for signs of damage
and necrosis. Measurement of liver nonprotein thiols (a pool comprised mainly of GSH
molecules) and liver total thiols served as measures of oxidative stress.

Knockout Mice. All mice had similar and normal serum ALT activity levels at 0
h (Fig. 4-3). At 12 h all mice had significantly elevated serum ALT activity, but the mean
ALT level (actual ALT units) was 6-12 times greater in KO mice than in CK mice. At 24
h and 48 h after CC14, the ALT levels had declined, but were still significantly greater in
CC14 treated mice. There were no genotype effects at the 24 h and 48 h points, and no
effects of dietary zinc at any time point.

Hematoxylin- and eosin-stained liver sections were similar in all groups injected
with corn oil (data not shown). Consistent with serum ALT activities, at 12 h there was
significantly more liver necrosis in KO mice (Fig. 4-4B) compared to CK mice (Fig. 4-
4A). The perivenous regions of the KO liver sections displayed coagulation necrosis.
Cells were less eosinophilic, displayed a general loss of morphology, and many contained
pyknotic nuclei. Hepatic sinusoids were collapsed and the hepatic plate arrangement was
disrupted. In contrast, little necrosis was visible in CK liver sections. These differences
were not seen at 24 h and 48 h, however. There were no differences between dietary
groups.

Oxidative stress was measured as nonprotein thiol levels (Fig. 4-5 A). Nonprotein
thiols (NPT) are a thiol pool made up largely of glutathione, and have been shown to
decrease in instances of oxidative stress (Powers et al. 1998). Although glutathione

concentrations are subject to circadian rhythms, this variable was controlled for by
inclusion of corn oil-treated mice for all combinations of genotype and dietary zinc.
Interestingly, NPT levels at 0 h tended to be lower in Zn500 groups than Zn10 groups, but
the difference was not quite significant (p = 0.06). At 12 h post injection, there was a

Figure 4-4. Light micrographs (200X magnification) of hematoxylin- and eosin-stained
liver sections from metallothionein knockout (KO) or control (CK) mice 12 h after
injection of carbon tetrachloride (CC14). (A) CK mouse; (B) KO mouse. All mice were
fed diets contaoining 10 mg Zn/kg diet (Znl0). More severe coagulation necrosis was seen
around the central vein (cv = central vein) in KO mice (Panel B) compared to CK mice
(Panel A). Results were similar in female mice and mice fed diets containing 500 mg
Zn/kg diet (Zn500). No histological changes due to genotype or diet were noted in mice
given an injection of the corn oil vehicle. Bar = 100 urn.

general depression of NPT, but no differences among groups. At 24 h NPT levels were
significantly lower in the KO+CCl4 groups, while KO+CO groups returned to normal.
Interestingly, at 48 h CC14 treated mice have significantly more NPTs in liver than CO
treated mice. Total thiols (TT) are a measure of both protein and nonprotein thiols
combined, including the thiols of metallothionein. There was a trend toward greater TT
levels in KO mice than CK mice at 0 h (Fig. 4-5B). At 12 h post treatment, however,

Control

Knockout

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0 12 24 36 48
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Figure 4-5. Indices of liver thiol homeostasis and oxidative stress of metallothionein
knockout (KO) and control (CK) mice 0, 12, 24, or 48 h after injection of carbon
tetrachloride (CC14) or corn oil (CO). The mice had been fed either adequate dietary zinc
(Zn10) or supplemental dietary zinc (Zn500). (A) Liver nonprotein thiols (nmol/g liver)
were measured spectrophotometrically. Protein thiols had been removed by TCA
precipitation. (B) Liver total thiols (umol/g liver) were measured spectrophotometrically.
Data are presented as means ± SE of n = 3 (0 h) or n = 4-7 (12, 24, and 48 h) mice/group.
Statistical differences (p < 0.05) were determined by ANOVA for a three way factorial
design, followed by the Tukey-Kramer multiple comparison test.

liver TT levels in KO mice drop, and are significantly lower than in CK mice. At 24 h
and 48 h no differences were detected.

Transgenic overexpressing mice. Serum ALT activity was similar and normal at
0 h in all groups (Fig. 4-6). Serum ALT rose sharply in all groups treated with CC14 at 12
h, and remained elevated through 48 h. There were no differences due to genotype. ALT
was slightly greater in Zn500 treated mice than Zn10 treated mice at 48 h, however. Of
note, ALT activities do not return to basal levels, as was observed with CK and KO mice
(Fig. 4-3)

Histological analysis of hematoxylin- and eosin-stained liver sections revealed
significant necrosis in all CCl4-treated mice, but there were no significant differences
between genotype (Fig. 4-7 A,B). There was significant lymphocyte infiltration in the

Control

Transgenic

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Figure 4-6. Serum alanine aminotransferase activity of metallothionein transgenic
(TG) and control (CT) mice 0, 12, 24, or 48 h after injection of carbon tetrachloride
(CC14) or corn oil (CO). The mice had been fed either adequate dietary zinc (Zn10) or
supplemental dietary zinc (Zn500). These activities were measured
spectrophotometrically and expressed as log activity units/mL serum. Data are
presented as means ± SE of three (0 h) or 4-7 mice/group (12, 24, and 48 h). Data are
presented as means ± SE of n = 3 (0 h) or n = 4-7 (12, 24, and 48 h) mice/group.
Statistical differences (p < 0.05) were determined by ANOVA for a three way
factorial design, followed by the Tukey-Kramer multiple comparison test.

.cv-1

•

C-

' ■ " .-t CV .' .

»•• "

• -

Figure 4-7. Light micrographs (200X magnification) of hematoxylin- and eosin-stained-
liver sections from metallothionein transgenic (TG) or control (CT) mice 12 h after
injection of carbon tetrachloride (CC14). (A) CT mouse fed the Znl0 diet; (B) TG mouse
fed the ZnI0 diet; (C) CT mouse fed the Zn500 diet; (D) TG mouse fed the Zn500 diet; (E)
enlargement of the perivenous region from figure 4-7D. Significant lymphocyte
infiltration is seen in the area directly surrounding the central vein (cv) of mice of both
genotypes fed the Zn500 diet after receiving CC14 (C-E). Results were similar in female
mice. No histological changes were related to genotype were noted in mcie given an
injection of corn oil vehicle and fed either diet. Bar = lOOum (A-D) or 25 urn (E).

area directly surrounding the central vein of ce of both genotypes fed the Zn500 diet (Fig.
4-7C-E).

There were no significant differences in NPT between TG and CT mouse livers at
0 h (Fig. 4-8A). Twelve hours after injection, the NPT levels were higher in CCl4-treated
mice. This relationship reversed at 24 h, and NPT levels were greatest in CT+CO mice.
Similar results were seen at 48 h.

Total thiol levels were similar in all mouse groups at 0 h (Fig. 4-8B). At 12 h
there is a trend toward greater TT levels in TG500+CC14 mice compared to others. This is
likely due to induction of hepatic metallothionein. At 24 h, CCl4-treated mice had
significantly greater TT levels than CO treated mice. Also, TG mice had significantly
greater TT levels than CT mice. These significances disappeared at 48 h, but there is still
a trend toward greater TT levels in TGCCW mice compared to TGco mice. Since the thiols
of metallothionein would be included in the TT measurement, and metallothionein
induction was greatest at 24 h and 48 h, metallothionein induction alone may have
accounted for the increased TT in TG mice at these time points. These also are the time
points of NPT suppression (Fig. 4-8 A).

Discussion

We examined the effects of supplemental dietary zinc in combination with
different levels of metallothionein gene expression on susceptibility to oxidative stress in
vivo. Previous research showed that supplemental zinc protected rat hepatocytes cultures
from various cytotoxic agents (Coppen et al. 1988, Schroeder and Cousins 1990). Zinc
treatment increased metallothionein gene expression, and it was suggested that
metallothionein protein was the mediator of the protection. Similar results were seen

when metallothionein expression was elevated by interleukin-6 and dexamethasone
(Schroeder and Cousins 1990). In this experiment, we used KO and TG mice as models
of different levels of metallothionein gene expression (Masters et al. 1994, Palmiter et al.
1993). By using two zinc intake levels, we were able to generate different hepatic zinc

Control

450

300

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Figure 4-8. Indices of liver thiol homeostasis and oxidative stress of metallothionein
transgenic (TG) and control (CT) mice 0, 12, 24, or 48 h after injection of carbon
tetrachloride (CC14) or corn oil (CO). The mice had been fed either adequate dietary
zinc (Znl0) or supplemental dietary zinc (Zn500). (A) Liver nonprotein thiols (nmol/g
liver) were measured spectrophotometrically. Protein thiols had been removed by
TCA precipitation. (B) Liver total thiols (umol/g liver) were measured
spectrophotometrically. Data are presented as means ± SE of n = 3 (0 h) or n = 4-7
(12, 24, and 48 h) mice/group. Statistical differences (p < 0.05) were determined by
ANOVA for a three way factorial design, followed by the Tukey-Kramer multiple
comparison test.

and metallothionein levels to examine their effects on the toxicity of the hepatotoxin
CC14. CCI4 is metabolized to the trichloromethyl radical by the enzyme cytochrome
P4502E1 in the perivenous region of the liver lobule (McGregor and Lang 1996, Sipes et
al. 1990; Wong et al. 1998). As a result, the perivenous region is most affected by this
radical species, which causes lipid peroxidation and inactivation of enzymes such as the
cytochrome P450 enzymes (McGregor and Lang 1996).

In several studies zinc pretreatment protected against CCl4-induced hepatotoxicity
in vivo (Cagen and Klaassen 1979, Chvapil et al. 1973, Dhawan and Goel 1995, Liu et al.
1998). This effect is only seen when copious amounts of dietary zinc (1000 mg/kg diet)
or parenteral zinc are given, however. For example, consumption of diets containing 300
mg Zn/kg, twenty fold the requirement for the rat, did not protect against CCl4-induced
hepatotoxicity in rats (Khoo et al. 1996). A proposed mechanism for the protective effect
of pharmacological doses of zinc is that zinc induces metallothionein, and that
metallothionein is the real mediator of the hepatoprotection. Metallothionein has been
shown to covalently bind CC14 metabolites and decrease the amount of these metabolites
bound to other cellular proteins (as reviewed in Klaassen and Liu 1998). In this way
metallothionein may prevent CC14 from reaching some of its cellular targets. Although
this might explain some of the zinc related protection, large parenteral zinc doses also
protect against CC14 toxicity in the absence of metallothionein expression (Itoh et al.
1997). This might be related to suppression of CC14 bioactivation, since supplementation
has been shown to inhibit the activity of some cytochrome P450 enzymes (Bray et al.
1986, Coppenetal. 1988).

Since metallothionein expression has been inversely related to damage after an
oxidative insult, we expected that the damage in mouse livers in this experiment would
be inversely proportional to metallothionein expression (i.e., TG < CT and CK < KO). In
support of this, ehanced susceptibility of KO mice to CC14 toxicity was reported recently
(Liu et al. 1998B). Also, we assumed that mice consuming the Zn500 diet would be
protected compared to those eating the Znl0 diet since supplementation would induce
more metallothionein protein and result in greater cellular zinc accumulation. Finally,
since metallothionein expression was thought to be key to zinc-related cytoprotection, we
expected no protection against hepatotoxicity by zinc supplementation in KO mice.

In support of our hypothesis we saw greater hepatotoxicity in KO mice compared
to CK mice (6-12 fold higher ALT values) at the 12 h time point. At later time points,
however, toxicity seemed equivalent in these genotypes. This is in spite of the fact that
KO mice had no metallothionein present in liver and were unable to sequester additional
hepatic zinc. Also, TG and CT mice did not differ in the level of hepatotoxicity produced
despite huge differences in hepatic metallothionein and zinc. Further, the nonprotein thiol
levels were not dramatically altered during the experiment, similar to results from
experiments with acetominophen in KO mice (Liu et al. 1999 A).

To our knowledge, this is the first report where the effect of supplemental dietary
zinc on CCl4-induced hepatotoxicity in mice was examined. Also, this is the first
assessment of the combination of supplemental dietary zinc and toxicity of any kind in
metallothionein overexpressing mice and metallothionein knockout mice. We show for
the first time using these models that neither supplemental dietary zinc nor
metallothionein overexpression alone protected against CCl4-induced hepatotoxicity in

mice. Further, no combination of metallothionein gene expression and either adequate or
supplemental dietary zinc provided protection, even though the levels of liver zinc and
liver metallothionein varied over a large range among groups.

These results argue against a direct antioxidant role for MT against CC14 toxicity,
since antioxidant protection against hepatotoxicity would likely be dose dependent
(Tirmenstein et al. 1997, Yao et al. 1994). Instead, the data fit better in a plateau model,
where metallothionein expression was important up to a point (< the level in CT and CK
mice), but beyond this point further expression is not useful. This is more in line with a
metallothionein-specific function, such as regulation of tissue zinc accumulation and/or
intracellular zinc trafficking. Specifically, KO mice might be less protected against CC14
than CK mice because they are unable to regulate zinc homeostasis appropriately (Coyle
et al. 1995, Davis et al. 1998, Philcox et al. 1995).

The most marked differences between genotypes in this experiment were
alterations in zinc metabolism. In both the TG and the KO experiments, the animals with
the lowest metallothionein expression displayed the weakest control over serum zinc
levels 12 h after CC14 treatment. In fact, serum zinc rose in KO mice and declined in TG
mice. This is coincident with induction of hepatic metallothionein and elevation of
hepatic zinc in all but the KO mice at this time point. This is also coincident with greater
hepatotoxicity in KO mice.

Metal lothionein's role in maintaining appropriate hepatic zinc levels might be
especially important under conditions of stress, including oxidative stress.
Metallothionein is induced during the acute phase response and during hepatic
regeneration, and is required for normal hepatic regeneration after partial hepatectomy

(Arora et al. 1998, Ohtake 1978). Also, zinc can be mobilized from metallothionein by a
number of oxidants or shifts in glutathione redox status (Berendji et al. 1997, Fliss and
Menard 1992, Tatsumi and Fliss 1994). These may be mechanisms for mobilization of
intracellular zinc during oxidative stress (Maret 1995). The end result may be enhanced
transfer of zinc from metallothionein to zinc-dependent proteins (Jiang et al. 1998). Zinc
mobilization may also affect gene expression.

If metallothionein acts as a zinc-donating molecule during oxidative stress, KO
mice might not be able to keep up with the demand for zinc incorporation into zinc-
dependent proteins. As a result, the level of functioning zinc-dependent proteins
produced during oxidative stress may not be sufficient in KO mice, and may explain why
KO mice were more adversely affected than CK mice. On the other hand, TG mouse
cells have more zinc available for donation to zinc-dependent proteins, but they may not
be able to produce those proteins rapidly enough to take advantage of the excess zinc. In
this case, the level of MT protein produced in CT mice is both necessary and sufficient,
and TG mice would not be better protected.

While other explanations for the results of this experiment exist, we can rule out
several. First of all, the results of this experiment are not likely affected by differences in
other antioxidants in these mice since the levels of other antioxidant enzymes and
molecules are reported to be similar between genotypes (Iszard et al. 1995, Kang et al.

1997, Lazo et al. 1995, Liu et al. 1999, Rofe et al. 1998, Wang et al. 1999, Wu and Kang

1998, Zheng et al. 1996). Also, bioactivation of CC14 should be similar between
genotypes since the activity of cytochrome P4502E1 is similar in TG and KO mouse
livers to their respective controls (Iszard et al. 1995, Itoh et al. 1997, Rofe et al. 1998).

Since these results are in opposition to some experiments done in rats, we cannot exclude
species difference as a confounding variable. However, it seems unlikely that a process
as basic as radical scavenging (antioxidant action), a simple oxidation-reduction reaction,
would differ between two rodent species. It should be noted that different strains of mice
were used in these experiments, and, therefore, the results are not likely due to
peculiarities of any individual inbred strain. However, we cannot rule out the possibility
that knockout mice are better protected than we expected due to some adaptive
mechanism(s). For instance, altered gene expression has been reported in KO mice
(Kimura et al. 2000).

Two other research groups have published reports of CCl4-induced hepatotoxicity
in KO mice, but no reports have been forwarded for TG mice (Itoh et al. 1997, Liu et al.
1998B). Those KO studies were designed to differentiate the hepatoprotective effects of
exogenous compounds (sakuraso-saponin and oleanic acid) from their ability to induce
metallothionein synthesis. Both groups used the same KO mouse model, injected 50 ul
CCl4/kg bw, and reported results for 24 h post-dose. Itoh and coworkers found no
difference in hepatotoxicity between genotypes as measured by plasma GOT activity. Liu
and coworkers found greater damage in KO mice as measured by serum ALT, serum
SDH, and histological analysis of hematoxylin- and eosin-stained liver sections. Itoh and
coworkers injected the dose subcutaneously, while Liu and coworkers injected the dose
intraperitonealy. Slower uptake of CC14 from the subcutaneous injections of Itoh and
coworkers might explain why no toxicity was seen at the 24h time course in their study.
Serum ALT values and histological analysis from the 12 h time point in this report match
well with the results of Liu and coworkers at 24 h. The differences in the time course of

hepatotoxcity between that experiment and ours may have been due to the smaller dose
(20 ul CCl4/kg bw) used in our experiments.

The lack of protection against oxidative stress by MT overexpression in this
experiment is in line with results from several other experiments that used models of
metallothionein gene overexpression. Early studies using transfection of the human MT-
2a gene into Chinese hampster ovary Kl-2 cells and several of tumor cells lines found no
resistance against free radical generating agents (Kaina et al. 1990, Kelley et al. 1988).
Further, TG mice were not resistant to adriamycin cardiotoxicity or y-irradiation
(DiSilvestro et al. 1996, Liu et al. 1999B). The existing evidence that metallothionein
overexpression alone protects against oxidative stress was found in transformed cells and
sheep pulmonary artery endothelial cells (SPAEC) transfected with metallothionein, and
with a second TG mouse strain that only overexpresses metallothionein in the heart. In
the former case, transfection of NIH 3T3 cells with MT-1 protected against nitric oxide
induced cytotoxicity (Schwarz et al. 1995). Viable cell determinations were not made
until 6 to 7 days after nitric oxide treatment, however, so it could be argued that the
difference in the number of cells remaining at that time was due to improved cell
recovery instead of radical scavenging. Protection against hyperoxia and tertiary-butyl
hydroperoxide was also seen in SPAEC transfected with mouse or human MT genes, but
the determination of viable cell numbers were not performed until 1-2 days after oxidant
exposure was initiated (Pitt et al. 1997). Again, it is difficult to separate the contributions
of antioxidation and cell recovery to cell survivial.

In the case of the heart-specific metallothionein overexpressing TG mouse strain,
there is both in vivo and in vitro evidence that metallothionein protected against

oxidative stress, including ischemia reperfusion, hydrogen peroxide, and doxorubicin
treatment (Kang et al. 1997, Kang et al. 1999, Wang et al. 1999, Wu and Kang 1998).
While this provides convincing evidence for an antioxidant role for metallothionein in
cardioprotection, it should be noted that a very high level of overexpression (10-130-
fold) is needed to see these effects, as lower levels of overexpression (3-fold) didn't
protect against adriamycin cardiotoxicity (DiSilvestro et al 1996). Further, mouse heart,
an organ that metallothionein is not normally abundant in, has a much weaker antioxidant
capacity than mouse liver (as reviewed in Kang 1999). So while this model confirms that
metallothionein overexpression can protect mouse heart against oxidative stress, the
effects may be small since they were unmasked only when metallothionein expression
was astronomically high, in an organ that doesn't normally produce much
metallothionein, yet is highly susceptible to oxidative stress. Therefore, it cannot be
assumed that the same results would be seen in the liver.

The lack of protection against CC14 hepatotoxicity by zinc supplementation (500
mg/kg diet) in any of the genotypes used in this experiment strongly suggests that the
required dietary zinc level for mice (10 mg/kg diet) provides as much protection as is
possible by dietary zinc. It also suggests that the hepatoprotective effects associated with
zinc injection are not readily reproduced with dietary zinc. Combined with data from zinc
deficiency studies we see a plateau affect of dietary zinc against oxidative stress in the
rodent model, just as we do with metallothionein. Zinc-deficient diets render rodents
more susceptible to oxidative stress, zinc-adequate diets alleviate this condition, but
supplemental dietary zinc provides no further protection.

The results of the experiments explained above confirm the importance of
metallothionein expression in protection against oxidative stress, but bring into question
the impact of supplemental zinc and/or elevated metallothionein expression in defense
against oxidative stress in mouse liver in vivo. Further, the protection against oxidative
stress appears to correlate with changes in zinc metabolism.

CHAPTER 5

EFFECTS OF METALLOTHIONEIN GENE EXPRESSION AND SUPPLEMENTAL

ZINC IN PROTECTION AGAINST OXIDATIVE STRESS IN PRIMARY

HEPATOCYTE CULTURES FROM METALLOTHIONEIN TRANSGENIC AND

METALLOTHIONEIN KNOCKOUT MICE

Introduction

As indicated in the previous chapter, metallothionein and zinc are implicated in
cellular antioxidant defense. In the previous experiment with metallothionein knockout
mice (KO) we found that metallothionein expression protected against carbon
tetrachloride-induced oxidative stress, but metallothionein overexpression in transgenic
mice (TG) did not provide further protection. Further, mice fed diets with supplemental
zinc (500 mg Zn/kg diet) were not protected compared to mice fed only the required zinc
intake (10 mg Zn/kg diet). These results are inconsistent with direct antioxidant activity
of metallothionein induction or supplemental zinc. To more directly assess the cellular
antioxidant functions of supplemental zinc and metallothionein, We studied the effects of
metallothionein expression and moderate zinc supplementation on tertiary-butyl
hydroperoxide-induced cytotoxicity in primary hepatocyte cultures from TG and KO
mice. Specifically, hepatocytes from metallothionein knockout mice can be used to
determine if zinc acts in cytoprotection independent of metallothionein by examining
whether these cells had altered sensitivity to tertiary-butyl hydroperoxide. We also
determined whether treating cells with zinc and/or dexamethasone and interleukin-6, all
of which induce metallothionein expression, influences sensitivity to tertiary-butyl

hydroperoxide in these genotypes. The results of this study provide further evidence that
metallothionein and supplemental zinc do not act directly as antioxidants.

Materials and Methods
Animal Model

Metallothionein knockout and metallothionein transgenic mice used in this study
were bred in house using founder mice purchased from The Jackson Laboratory, Bar
Harbor, ME. The metallothionein overexpressing mice (designated TG mice) were
originally generated in C57BL/6 mice crossed with SJL mice (Palmiter et al. 1993).
Backcrossing against C57BL/6 mice permit the use of C57BL/6 mice as controls
(designated CT mice). The metallothionein knockout mice (designated KO mice) were
generated in 129/SvCPJ mice crossed with C57BL/6 mice (Masters et al. 1994). These
mice were maintained on a 129/SvCPJ background. 129S3/SvImJ mice served as controls
(designated CK mice). All experiments used 7-11 wk old female mice. Mice were housed
in plastic box cages with a 12 h light:dark cycle. The mice were given access to tap water
and a standard rodent diet (Harlan Teklad 8604, Madison, WI) until they were used for
hepatocyte preparations. Care and treatment of the mice received approval of the
University of Florida Institutional Care and Use Committee.

Hepatocyte Isolation

All liver perfusions began between 8 AM and noon. In all experiments
hepatocytes were collected from mice of both genotypes (KO and CK or TG and CT) on
the same day. Mice were anaesthetized with sodium pentabarbital (60 mg/kg i.p.).
Hepatocytes were isolated using a two-step perfusion technique run retrograde from the

inferior vena cava, with perfusate allowed to flow out of the portal vein without
recirculation (Schroeder and Cousins 1991, Renton et al. 1978). Livers were first
perfused with a Ca-free buffered solution (8-10 mL/min), followed by a buffered solution
containing collagenase. After perfusion the liver was aseptically transferred to a sterile
cell culture hood. The liver was disrupted and the cells were liberated using a cell
scraper.

Hepatocyte Culture

The hepatocytes were suspended in wash medium (Williams Medium E with 10
mmol/L HEPES buffer and 10 mmol/L TES buffer) followed by centrifugation (50 x g
for 3 min) to separate viable cells from debris. The wash and centrifugation steps were
repeated two times. The final cell pellet was resuspended in culture medium (Williams
Medium E supplemented with 10% FBS, 100 nmol/L insulin, 100 units/mL penicillin,
100 ug/mL streptomycin). Cell number and viability were determined using a
hemacytometer and trypan blue exclusion. Preparations with greater than 85% viability
were used for experiments. Cells were added to type I collagen-coated 35 mm tissue
culture plates (0.5x 106 cells/plate in 2 mL medium) and allowed to attach (37°C, 5%
C02). After a 2-3 h attachment period the culture medium was removed and replaced
with 1 mL of one of four different medium combinations: medium containing 4 umol
zinc/L, medium with added zinc (32 umol/L), medium with dexamethasone (1 umol/L)
and interleukin-6 (100 units/mL), and medium with added zinc, dexamethasone and
interleukin-6. Cells were maintained in these culture conditions for 1 8-22 h prior to
addition of tertiary-butyl hydroperoxide (TBH). Carbon tetrachloride was also used in

some experiments, but its nonpolar nature and volatility led to problems with
reproducibility within and between experiments.
Cytotoxicity assays

After the culture period was complete the medium was again removed and
replaced with medium containing 0 to 500 umol/L TBH for 30 to 150 min. At the end of
TBH treatment, cells and medium were prepared for determination of lactate
dehydrogenase (LDH) activity. Briefly, medium was centrifuged (13000 x g) to remove
unattached cells and debris, and then stored at -20°C for up to 48 h before assay. Cells
were removed in 1% triton X-100, disrupted by repeated passage through 200 uL pipette
tips, and centrifuged (13000 x g). The supernatant was diluted with 4 volumes of Tris
buffer (100 mmol/L 2-amino-2-(hydroxymethyl)-l,3-propanediol, pH 7.5) and stored at -
20°C for up to 48 h before assay. LDH activity was assayed spectrophotometrically
using a commercial kit (Sigma LD-L) by incubating aliquots of medium or cell extracts
(125 uL) with reagent solution (375 uL) containing lactate (50 mmol/L) and NAD (7
mmol/L) in buffer (pH 8.9). LDH in the sample catalyzes the reaction of lactate and
NAD, resulting in the production of pyruvate and NADH. Formation of NADH results in
an increase in absorbance at 340 nm. The rate of increase in absorbance is directly
proportional to LDH activity in the sample. The percent LDH leakage is calculated as the
LDH activity of the medium as a percentage of the sum of the LDH activities of the
medium + the cells (Jauregui et al. 1981).

Cell viability was also determined by the ability of live cells to convert 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) to a colored
product (Denizot and Lang 1986). In these experiments, cells were cultured on collagen

coated 96 well plates (3 x 104 cells/well in 200 uL WME) and treated as described above
for 35 mm plates. At the end of the culture period the medium was replaced with medium
containing 1-50 umol/L TBH and incubated for 120 min. This lower range of TBH
concentrations was used in these experiments to correct for the greater ratio of medium
volume-to-cell number. Medium containing TBH was then replaced with serum-free
culture medium containing MTT (2.4 umol/L) for 3 h. MTT is a tetrazolium dye that is
converted to an insoluble purple formazan through cleavage of the tetrazolium ring by
dehydrogenase enzymes of live cells. Cultures were washed and then solubilized in 100
uL acidic isopropanol (40 umol/L HC1 in absolute isopropanol). After shaking the wells
were analyzed (A560-A650). Data are presented as percent of activity of untreated cultures.

Analytical Methods

For intracellular glutathione analysis cells were homogenized in 1% (39 mmol/L)
sulfosalicylic acid (2-hydroxy-5-sulfobenzoic acid; SSA) and placed on ice (> 20 min) to
precipitate proteins, which were then removed by centrifugation (13000 x g). The
supernatants were frozen overnight (-20°C). Total glutathione (oxidized + reduced) was
measured spectrophotometrically by the glutathione (GSH) reductase-recycling assay
using a microtiter plate reader (Baker et al. 1990). Briefly, supernatants were diluted with
buffer (100 mmol/L sodium phosphate, 1 mmol/L EDTA, pH 7.5) and reacted with 5,5'-
dithiobis-(2-nitrobenzoic acid) (DTNB; 150 umol/L) in the presence of NADPH (100
umol/L) and GSH reductase (1.0 units/mL). Reaction of GSH with DTNB generates
oxidized glutathione (GSSG) and the highly colored 5-thio-2-nitrobenzoic acid anion,
which can be measured at A. = 410 nm. GSH is regenerated from GSSG by glutathione
reductase (using reducing equivalents from NADPH), which allows the reaction between

GSH and DTNB to continue at a linear rate. GSH concentration is determined from a
standard curve using GSSG. 109Cd (1.35 GBq/nmol; Isotope Product Laboratories,
Burbank, CA) was measured using a Packard Cobra II gamma spectrometer (Packard,
Downers Grove, IL). Metallothionein protein content of the cells was measured prior to
treatment with oxidant by the cadmium (109Cd) binding assay (Eaton and Toal 1982).
Briefly, cells were lysed in 10 mmol/L Tris buffer containing protease inhibitors (0.1
mmol/L phenylmethylsulfonylfluoride, 1.2 u,mol/L leupeptin and 1.5 umol/L pepstatin
A). Homogenates were centrifuged (10000 x g), the supernatant was boiled and
centrifuged again (10000 x g), and the resulting supernatant was incubated with 109Cd.
Unbound l09Cd was removed using hemoglobin. 109Cd bound to metallothionein was
measured by y-counting, and converted to moles of metallothionein (Davis et al. 1998).
Cell zinc was measured by atomic absorption spectrophotometry after cells were
solubilized in an aqueous solution of sodium dodecyl sulfate (7 umol/L) and sodium
hydroxide (400 mmol/L) as previously described (Schroeder and Cousins 1991). Total
protein was measured by the method of Lowry et al. (1951).

Statistics

Data were analyzed by ANOVA for a three way factorial design (2x2x2) to
determine significant main effects and interactions among genotype, medium zinc and
dexamethasone/Il-6 treatment (SAS, SAS Institute Inc. Cary, North Carolina). The
Tukey-Kramer post hoc test was used to determine significant differences between
specific groups when interactions were significant (p < 0.05).

Results

Hepatocyte metallothionein expression was directly related to MT genotype and
was inducible by zinc treatment, dexamethasone and 11-6 (dex/Il-6) treatment, and the
combination of these treatments in all but KO cells (Fig. 5-1). Also, induction by these
treatments was greater in TG cells than either control strain. Further, metallothionein
induction by the combination of zinc treatment and dex/Il-6 treatment was synergistic.

Cell zinc concentrations were lower in KO cultures than CK cultures under all
treatment conditions (Table 5-1). As with metallothionein, cell zinc was increased in CK
cultures in response to zinc treatment and dex/Il-6 treatment, and was increased
synergistically by these two treatments combined. Cell zinc was not affected by these
treatments in KO cultures, however.

Lactate dehydrogenase (LDH) enzyme leakage was used as a measure of
cytotoxicity. Leakage was not greatly different between CK and KO cultures after
treatment with TBH (350 umol/L or 450 umol/L) when cells were not previously
exposed to MT inducers (Fig. 5-2A). Zinc pretreatment modestly elevated LDH
leakagefrom CK mouse hepatocytes treated with 450 umol/L TBH, but did not alter
toxicity in KO cultures. Treatment with dex/Il-6 slightly increased LDH leakage in KO
cells in response to 450 umol/L TBH, but increased toxicity in CK cells by three fold. It
should be noted that neither zinc treatment nor dex/Il-6 treatment increased LDH leakage
in the absence of TBH. The combination of zinc treatment with dex/Il-6 treatment was
associated with a similar level of TBH- induced toxicity as dex/IL-6 treatment alone.

Similar trends were seen in experiments with hepatocytes from TG and CT mice.
(Fig. 5-2B). LDH leakage was already greater in TG cultures than CT cultures without

exposure to metallothionein-inducing agents. Pretreatment with zinc slightly elevated
LDH leakage in CT cultures when exposed to 450 umol/L TBH, but nearly doubled
leakage from TG cells. Treatment with dex/Il-6 enhanced leakage even further in both
genotypes, but again the increase was greatest in TG cultures. Toxicity was 5-fold greater
in TG cultures pretreated with dex/Il-6 and exposed to 350 umol/L TBH than identically
treated CT cultures. Similar to the KO experiment, the combination of zinc treatment and
dex/Il-6 treatment resulted in TBH-induced toxicity similar to that seen with dex/Il-6
treatment alone.

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4000
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3000
2500
2000
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Figure 5-1. Metallothionein content of metallothionein knockout (KO), knockout control
(CK), metallothionein transgenic (TG), and transgenic control (CT) mouse hepatocytes.
Cells were cultured in Williams Medium E (WME) containing either 4 umol zinc/L
(control), 32 umol Zn/L (Zn), 1 umol dexamethasone/L and 100 units Il-6/mL (Dex/Il-6),
or 32 umol Zn/L, 1 umol dexamethasone/L and 100 units Il-6/mL (Zn + Dex/Il-6) for 20
h prior to harvest. MT was quantified for KO hepatocytes cultured in control or zinc
medium only. Data are means ± SEM (n = 4-8 cultures). Significant differences (p <
0.05) were determined using ANOVA for a 2x2x2 factorial design, followed by the
Tukey-Kramer multiple comparison test.

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Cell viability measured with the MTT assay revealed results similar to
those seen with the LDH assay. Viability was greater in untreated or zinc-treated KO

-
5

30
25
20
15

10
5
0

100
90
80
70
60
50
40
30
20
10
0

TBH (nmol/L)
■ 0
E2 350
□ 450

b b
I

a,b

iilili

a,b
X

<? <r <f

A- 4? J3 v-to

& <r <f

TBH (nmol/L)
■ 0
0 350
D 450

£~ </ </

a <i? Jp Jp

& >*

Figure 5-2. Tertiary-butyl hydroperoxide-induced cytotoxicity. Cytotoxicity was
measured as Lactate dehydrogenase leakage from metallothionein knockout (KO),
knockout control (CK), metallothionein transgenic (TG), and transgenic control (CT)
mouse hepatocytes after treatment with tertiary-butyl hydroperoxide (TBH). Hepatocytes
were cultured as in figure 5-1 for 20 h, then treated with 0, 350, or 450 umol/L TBH for
2 h. (A) LDH leakage from CK and KO mouse hepatocytes. (B) LDH leakage from CT
and TG mouse hepatocytes. Data are means + SEM (n ■ 5-6 cultures from a
representative experiment). Significant differences (p < 0.05) between groups treated
with TBH were determined using ANOVA for a 2x2x2 factorial design, followed by the
Tukey-Kramer multiple comparison test.

cultures than CK cultures when exposed to 10 umol/L TBH, and in all KO cultures
compared to CK cultures at 15 umol/L TBH (Table 5-2). All TG cultures had lower
viability than CT cultures at 10 umol/L TBH (Table 5-3). Nearly all cells from these
genotypes were dead at 15 umol/L TBH.

Total glutathione was measured after the culture period but before the addition of
TBH to determine if glutathione status was affected by genotype, medium zinc and/or
dex/H-6 treatment (Table 5-4). Glutathione concentrations were 25-55% lower in CK
cultures than KO cultures, depending on the culture condition. Dex/Il-6 treatment
reduced cellular GSH in both genotypes, but to much lower levels in CK cultures. Zinc
treatment alone, or in combination with dex/Il-6 had little affect on glutathione levels.

Discussion

Previous work from this lab found that zinc supplementation protected against
oxidative stress in primary cultures of rat hepatocytes (Coppen et al. 1988, Schroeder and
Cousins 1990). Protection was credited, in part, to metallothionein induction by zinc.
Induction of metallothionein with hormones and cytokines was also correlated with such
protection (Schroeder and Cousins 1990). We recently found that metallothionein
expression protected mice against carbon tetrachloride heptotoxicity, but neither
supplemental dietary zinc nor metallothionein overexpression provided further protection
in metallothionein knockout and metallothionein transgenic mice (Davis et al.
submitted). These results were inconsistent with general antioxidant functions for
metallothionein and supplemental dietary zinc, and are in opposition to a number of
previously published reports (Liu et al. 1998 A, Liu et al. 1999A, Rofe et al. 1998). With

Table 5-2. Cell viability after tertiary-butyl hydroperoxide exposure of hepatocyte
cultures from metallothionein knockout and control mice previously treated with zinc
and/or dexamethasone and Il-6abc

Cell Viability (% of untreated cultures)

ANOVA Results

Genotyp*

i TBH Control

Dex/Il-6 Zn &

umol/L

Dex/Il-6

p value

CKd

5 61 ±2

65 ±2

63 ± 2 82 ± 1

KOe

84 ±1

70 ±1

62 ± 2 63 ± 2
Genotype x Zinc x Dex/Il-6
Genotype x Zinc
Genotype x Dex/Il-6
Zinc x Dex/Il-6
Genotype
Zinc
Dex/Il-6

0.8090
0.0001
0.0001
0.0001
0.0625
0.0741
0.0655

10 54 ±3

52 ±2

66 ± 2 72 ± 2

90 ±4

73 ±3

66 ± 3 62 ± 2
Genotype x Zinc x Dex/Il-6
Genotype x Zinc
Genotype x Dex/Il-6
Zinc x Dex/Il-6
Genotype
Zinc
Dex/Il-6

0.4920
0.0020
0.0001
0.0030
0.0001
0.0435
0.8038

15 11±1

9±1

12±1 18±1

32 ±2

28 ±1

27 ± 2 26 ± 2
Genotype x Zinc x Dex/Il-6

0.5875

Genotype x Zinc

Genotype x Dex/Il-6

Zinc x Dex/Il-6

Genotype

Zinc

Dex/Il-6

0.0380
0.0016
0.0063
0.0001
0.7010
0.5909

a Cell viability values are means ± SEM, n = 10-11.
b Zinc treatment was 32 umol/L for 20 h.

c Dexamethasone and 11-6 treatment (Dex/Il-6) was 1 umol/L Dex and 100 units Il-6/mL
for 20 h.

CK, hepatocyte cultures from control mice.
e KO, hepatocyte cultures from metallothionein knockout mice.

the experiments described herein we attempted to determine whether metallothionein
induction and supplemental zinc could act as cellular antioxidants against TBH-induced
toxicity.

We used primary hepatocytes cultures from TG and KO mice as a cellular model
of stably altered metallothionein expression, independent of metallothionein inducing
agents. In addition we assessed the effects of zinc and/or dexamethasone and IL-6
treatment on cytotoxicity in these cells, which have inherently different abilities to
produce metallothionein. In these experiments we found a consistent, direct relationship
between cellular metallothionein content and susceptibility to TBH toxicity. This
relationship occurred whether metallothionein was elevated due to genotype, inducing
agents (zinc or dexamethasone and 11-6 treatment), or their combination. Further,
dexamethasone and 11-6, powerful metallothionein inducers and protectors against
hepatocyte toxicity in rats, increased sensitivity to TBH toxicity in these mice. To our
knowledge, this is the first report that demonstrates an inverse relationship between
metallothionein expression and susceptibility to an oxidative stress.

Although the results of this study were unexpected, possible explanations can be
proposed. Antioxidant defense against the toxicity of tertiary-butyl hydroperoxide
involves consumption of considerable quantities of glutathione (GSH) by the antioxidant
enzymes glutathione peroxidase and phospholipid glutathione peroxidase (Rush et al.
1985). In addition, glutathione transferase enzymes may detoxify genotoxic products of
lipid peroxidation by conjugatng them to glutathione (Hubatsch et al. 1998). As such,
disturbances in GSH metabolism may enhance TBH toxicity. For example, hepatocyte
glutathione levels decline when cysteine availability is low (Wang et al. 1997). In our

Table 5-3. Cell viability after tertiary-butyl hydroperoxide exposure of hepatocyte
cultures from metallothionein transgenic and control mice previously treated with zinc
and/or dexamethasone and Il-6a' c

Cell Viability (% of untreated cultures)

ANOVA Results

Genotype

: TBH Control

Dex/Il-6 Zn &

umol/L

Dex/Il-6

p value

CTd

5 82 ±5

79 ±2

78 ± 2 80 ± 3

TGe

76 ±6

84 ±5

94 ± 8 87 ± 5
Genotype x Zinc x Dex/Il-6
Genotype x Zinc
Genotype x Dex/Il-6
Zinc x Dex/Il-6
Genotype
Zinc
Dex/Il-6

0.1038
0.8780
0.1500
0.2906
0.1553
0.7956
0.3624

10 58±3X

48 ± 2x>y

53 ± 3x'y 48 ± 2x'y

48 ± ly

33±2Z

29±2Z 31±lz
Genotype x Zinc x Dex/Il-6
Genotype x Zinc
Genotype x Dex/Il-6
Zinc x Dex/Il-6
Genotype
Zinc
Dex/Il-6

0.0415
0.7802
0.0054
0.0007
0.0001
0.0001
0.0001

15 18±1

15±1

21 ±2 16±1

14±1

13 ±0

14 ±0 13 ±0
Genotype x Zinc x Dex/Il-6
Genotype x Zinc
Genotype x Dex/Il-6
Zinc x Dex/U-6
Genotype
Zinc
Dex/Il-6

0.5971
0.0204
0.0434
0.5886
0.0001
0.0021
0.1061

a Cell viability values are means ± SEM, n = 9- 1 1 .

b Zinc treatment was 32 umol/L for 20 h.

c Dexamethasone and 11-6 treatment (Dex/Il-6) was 1 umol/L Dex and 100 units Il-6/mL

for 20 h.

CT, hepatocyte cultures from control mice.
e TG, hepatocyte cultures from metallothionein transgenic mice.
x'y,z Means with different superscript values are significantly different.

experiments we found that as metallothionein accumulation increased, cellular
glutathione decreased (Table 4). Since cysteine residues make up 33% of the total amino
acid content of metallothionein protein, induction of MT protein may consume a
significant amount of the cellular cysteine pool. Consequently, induction of
metallothionein by dex/Il-6 treatment may have depleted glutathione levels and enhanced
TBH toxicity by competing for cysteine needed for GSH synthesis. Further, depletion of
cysteine pools might have inhibited the GSH regeneration during the toxic insult.
Alternatively, excess metallothionein may form mixed disulfides with oxidized
glutathione (GSSG) generated during TBH-induced oxidative stress (Brouwer et al. 1993,
Chai et al. 1994). Mixed disulfide formation may remove glutathione from its
regenerative pathway, delaying or preventing regeneration of reduced GSH needed for
antioxidant protection and other cellular processes (Gilbert 1995, Meister 1995).

The reduced sensitivity of KO cultures to TBH toxicity might also be explained if
there is a difference in the peroxidizability of cellular membranes between genotypes.
For instance, testes of zinc-deficient rats have a lower peroxidizable fatty acid level, and
are resistant to peroxidation in vitro (Oteiza and Keen 1996). If cells from KO mice act
similar to zinc-deficient tissues due to the lack of the intracellular ZnMT pool, they may
also have lower levels of the most peroxidizable fatty acids, and gain resistance to TBH
toxicity.

Several cell culture experiments have shown that treatment with metallothionein-
inducing agents is associated with protection against oxidants (Coppen et al. 1988,
Schroeder et al. 1990). Similar results were found in cells transfected with
metallothionein genes (Pitt et al. 1997, Schwarz et al. 1995) or cells from TG and KO

mice (Lazo et al. 1995, Rofe et al. 1998, Wang et al. 1999, Zheng et al. 1996). Of the two
studies that used primary hepatocyte cultures from KO mice, one showed only modest
protection against TBH (Zheng et al. 1996). The other report showed significant
protection by metallothionein expression against paracetamol toxicity, but only when
cultures were derived from fed mice; no differences in toxicity were seen between
genotypes in cultures from fasted mice (Rofe et al. 1998). Further, none of those studies
investigated whether inducing metallothionein prior to oxidant treatment affects toxicity.
Several other studies failed to find any protection by metallothionein expression (up to
166-fold) against the free radicals produced by x-radiation, bleomycin, or doxorubicin

Table 5-4. Cellular glutathione concentrations in hepatocyte cultures from metallothionein
knockout and control mice after treatment with zinc or dexamethasone and Il-6abc

Cell glutathione (umol/g protein)

ANOVA

Genotype

Control

Zn Dex/Il-6 Zn & Dex/Il-6

p value

CKd

23 ±3

24 ± 2 1 1 ± 1 1 1 ± 1

KOe

31±3

40 ± 2 25 ± 3 23 ± 2

Genotype x Zinc x Dex/Il-6

0.1066

Genotype x Zinc

0.3360

Genotype x Dex/Il-6

0.7097

Zinc x Dex/Il-6

0.0605

Genotype

0.0001

Zinc

0.1832

Dex/Il-6

0.0001

a Cellular glutathione values are means ± SEM, n = 6.

b Zinc treatment was 32 umol/L for 20 h.

c Dexamethasone and 11-6 treatment (Dex/Il-6) was 1 umol/L Dex and 100 units Il-6/mL

for 20 h.

d CK, hepatocyte cultures from control mice.

e KO, hepatocyte cultures from metallothionein knockout mice.

treatment (Kaina et al 1990, Kelley et al. 1988). Instead, they found protection to be more
consistent against alkylating agents. Our results contribute further evidence against a
cellular free radical scavenging function for metallothionein.

Supplemental zinc (32 umol/L) did not protect against oxidative damage in these
experiments. In fact, zinc treatment resulted in a consistent increase in LDH leakage in
all genotypes (Fig. 5-2). Other studies using mouse hepatocytes also found no protection
by zinc treatment (50 umol/L and 100 umol/L) against free radical generators (Tezuka et
al. 1995, Rofe et al. 1998). This is in contrast to several studies using zinc-supplemented
(48 umol/L) rat hepatocytes (Coppen et al. 1988, Schroeder et al. 1990). Zinc induced
metallothionein in rat and mouse hepatocytes, but zinc only protected rat cultures. Since
rat hepatocytes were protected and mouse hepatocytes were not, we cannot rule out
species difference as a confounding variable in these experiments. For example, mouse
hepatocytes are significantly more sensitive to TBH-induced toxicity than rats, possibly
due to a greater peroxidizable lipid content of cellular membranes (Rush et al. 1985).
Since GSH is an important substrate for detoxification of peroxidized lipids by
phospholipid glutathione peroxidase and glutathione transferase enzymes, hepatocytes
from mice may be more sensitive to glutathione depression than hepatocytes from rats if
lipid peroxidation is greater in mice after TBH treatment. It seems unlikely, however,
that chemical properties as basic as free radical scavenging (a simple oxidation-reduction
reaction) and zinc-thiol binding would differ between two rodent species. It should also
be noted that hepatocytes from two different mouse strains were used in our experiments.
As such, it is unlikely that the results presented here were due to peculiarities of any
particular mouse strain. Further, levels of other antioxidants do not differ among these

mouse genotypes (Iszard et al. 1995, Lazo et al. 1995). Instead, these results show that
supplemental zinc does not provide consistent protection against oxidative stress, and in
this case exacerbated toxicity.

Another interesting result was that dex/Il -6 treatment, which protected rat
hepatocytes cultures from oxidative stress (Schroeder et al. 1990), enhanced
susceptibility to TBH in both mouse strains in this experiment. The enhanced
susceptibility was closely related to the magnitude of metallothionein expressed after that
treatment, and was not altered by zinc-treatment. This is in opposition to the report of
Rofe and coworkers (1998), who found that dexamethasone (1 umol/L) protected CK
mouse hepatocytes against paracetamol toxicity when administered at the same time as
paracetamol. KO hepatocytes were not protected by dexamethasone treatment,
suggesting that metallothionein production was necessary for that effect. Metallothionein
was not induced before paracetamol exposure, however, which may explain why their
results were different than ours. In support of this, glutathione levels were similar
between genotypes before paracetamol exposure in their experiments. They also found
that supplemental zinc nearly tripled metallothionein levels in dexamethasone treated CK
cultures, yet inhibited dexamethasone-mediated protection against paracetamol (Rofe et
al. 1998). This suggests that overproduction of metallothionein may be counterproductive
even when metallothionein induction occurs during paracetamol exposure.

In conclusion, these results argue that supplemental zinc and/or metallothionein
do not protect hepatocyte cultures against tertiary-butyl hydroperoxide-induced oxidative
stress, but rather enhance the toxicity. Further, preinduction of metallothionein enhanced
the toxicity. Further research is required to determine the mechanism involved, but it is

likely related to depressed glutathione levels in hepatocytes after metallothionein
induction. If so, induction of metallothionein prior to oxidant exposure may be
counterproductive if protection against that oxidant relies on glutathione availability.

CHAPTER 6
CONCLUSIONS

The research reports bound together in this dissertation provide new insights into
the biochemical and physiological functions of metallothionein and zinc. The foci of this
research fall into two categories:

(1) the effects of metallothionein expression and dietary zinc intake in zinc
absorption, distribution, and intracellular zinc trafficking in TG and KO mice,

and

(2) the effects of supplemental zinc, metallothionein expression, and their
combination in defense against oxidative stress.

The discussion that follows brings together data from the four separate research
reports in an effort to provide a unified model of the biological roles of metallothionein
and zinc, and their interaction.

Zinc and Metallothionein in Zinc Absorption and Metabolism

Metallothionein Expression and Zinc Absorption

The results outlined in Chapter 2 provide further evidence for the theory that
induction of intestinal metallothionein is a least part of a mechanism for controlling the
flux of zinc from the intestinal lumen to the general circulation. Specifically, serum zinc
was elevated to a greater level in mice with lower levels of metallothionein expression
after an acute oral zinc dose (TG < CT & CK < KO). Similar results were found in a

separate metallothionein knockout strain over a range from normal to supplemental zinc
intake (Coyle et al. 1999, Coyle et al. 2000). Contrary to our hypothesis, however,
intestinal metallothionein induction did not result in greater zinc accumulation in the
intestine, suggesting that metallothionein 's role is not simply to sequester zinc in the
intestine. It may be that metallothionein acts to suppress zinc absorption by enhancing
zinc flux back toward the lumen, as suggested by Hoadley and coworkers (1988). It
should be stressed, however, that the effects of metallothionein on zinc absorption seen in
Chapter 2 can be interpreted only with reference to an acute oral dose of zinc.

Metallothionein Expression and Zinc Metabolism

The results of Chapters 3 through 5 provide insight into the overall impact of
metallothionein expression on zinc distribution, accumulation and trafficking at the
cellular and whole body levels. Data from Chapters 3 and 4 (only 0 h data from Chapter
4) display the effects of dietary zinc intake (ranging from 10 mg/kg diet to 500 mg/kg
diet) on metallothionein expression. Conversely, we also saw how the level of
metallothionein expression affects intestinal, hepatic and serum zinc concentrations.
Dietary zinc induced intestinal and hepatic metallothionein protein expression, but highly
supplemental dietary zinc intakes were required (> 200 mg/kg diet). At lower intakes
metallothionein was undetectable. Further, metallothionein was overexpressed in TG
mouse liver at all zinc intakes, yet did not affect hepatic zinc accumulation until diets
containing thirty-fold the requirement were consumed. At 500 mg Zn/kg diet, however,
hepatic zinc accumulation was directly related to hepatic metallothionein induction in all
genotypes. These results are directly in line with the role proposed for metallothionein in
zinc metabolism; i.e., metallothionein is required for zinc accumulation in tissues.

However, within the range of dietary zinc intakes that we studied, metallothionein
expression only altered tissue zinc accumulation under conditions of dietary zinc excess.
The situation might be very different under conditions of stress and zinc deficiency. For
example, Philcox and coworkers (2000) reported that metallothionein expression inhibits
intestinal zinc loss during stress (starvation and immune stress associated with
lipopolysaccharide injection), and prevents body zinc loss during zinc deficiency.

The results of Chapter 4 also show that hepatic zinc accumulation during stress is
dependent on metallothionein expression. This had been shown in a separate KO mouse
model after immune stress (Philcox et al. 1995). Our results extend this finding to the
level of metallothionein expression observed in TG mice consuming supplemental zinc
and subsequently treated with carbon tetrachloride. We also report that hepatic
metallothionein expression after exposure to stress was, in part, dependent upon the level
of zinc consumed in the diet. In Chapter 5 we report that intracellular zinc accumulation
in cultured hepatocytes after exposure to excess extracellular zinc, dexamethasone and
interleukin-6, and their combination was also dependent on the level of metallothionein
expression. This was also reported recently in another KO mouse model (Coyle et al.
1995).

Evidence was found implicating metallothionein in autoreguolation of its own
gene expression (Chapter 4). Metallothionein expression was stimulated at lower dietary
zinc intakes in mice with more copies of the metallothionein gene (TG > CT and CK >
KO). This finding could have larger implications, as it suggests that metallothionein
expression may also regulate expression of other zinc-responsive genes (Fig. 6-1). At this
time we speculate that metallothionein acts to regulate gene expression through one of

two mechanisms: metallothionein may act as a labile zinc pool that can release zinc for
use in zinc finger transcription factors (e.g., metal response transcription factor-1) that act
in upregulation of zinc-responsive genes, or metallothionein may directly donate zinc to
such proteins (Maret 2000, Zeng et al. 1991). Either way, these results suggest that
metallothionein may be involved in zinc metabolism at dietary zinc levels that do not
cause detectable changes in tissue zinc accumulation.

Before leaving this subject, it should be noted that although KO mice have altered
zinc metabolism, this is only seen under conditions of stress or supplemental zinc intake
(Chapters 2-5). For example, serum zinc levels are maintained accordingly in KO mice

Figure 6-1. Autoregulation of metallothionein gene expression. Metallothionein might
regulate expression of its own gene through one of two pathways: (A) zinc released from
ZnMT enhances the availability of free zinc for incorporation into transcription factors,
such as MTF-1, and/or (B) ZnMT directly donates zinc to MTF-1. Either scenario might
promote the DNA binding activity of MTF-1 by activating a key zinc finger within this
transcription factor protein.

allowed to acclimate for several days to diets ranging in zinc content from 10 to 200
mg/kg. In fact, serum zinc concentrations were lower in KO mice than CK mice
consuming diets with 300 and 500 mg Zn/kg. Obviously, KO mice can compensate for
the loss of metallothionein expression over a wide range of zinc intakes. Altered
expression of one or more zinc transporters may explain this adaptation response in KO
mice (Liuzzi et al. 2000, McMahon and Cousins 1998).

Zinc and Metallothionein in Defense Against Oxidative Stress

In Vivo

Metallothionein expression protected CK mice against carbon tetrachloride-
toxicity, confirming metallothionein' s role in protection against oxidative stress.
Metallothionein overexpression provided no further protection, however, with or without
added dietary zinc. Supplemental zinc alone was not protective either. These results are
inconsistent with a general (direct) antioxidant role for either zinc or metallothionein.
Instead, the result may be more indicative of the general need for rapid zinc accumulation
in the liver; a process that depends on metallothionein expression (chapters 3-5). We
speculate that the KO mice are more susceptible to oxidative stress because they lack the
capacity for rapid and sustained hepatic zinc accumulation. This zinc may be required for
numerous proteins (enzymes and transcription factors) involved in gene expression, as
well as other proteins involved in cellular metabolism. Since excess dietary zinc and
elevated metallothionein expression provide no additional protection, it may be that
adequate zinc intake and "normal" metallothionein expression are all that are needed to
maintain these processes. The liver may not be capable of synthesizing proteins quickly

enough to take advantage of the excess intracellular zinc provided via the diet or elevated
metallothionein levels. Alternatively, greater production of these proteins may not be
helpful.

In Vitro

Cytotoxicity studies in primary hepatocyte cultures from KO and TG mice were
undertaken to determine the protective roles of zinc and metallothionein against oxidative
stress in a simpler, more easily manipulated model. We found that induction of
metallothionein prior to tertiary-butyl hydroperoxide-treatment resulted in heightened
cytotoxicity; a result exactly opposite of that hypothesized, and opposite of what had
been reported previously with other oxidants. This relationship between metallothionein
expression and cytotoxicity was true whether metallothionein was elevated due to
genotype, zinc, dexamethasone and interleukin-6, and any combination of these factors.
Further, the magnitude of cytotoxicity was proportional to the magnitude of
metallothionein protein induced.

We found that metallothionein expression was inversely related to cellular
glutathione levels. Since the first line of defense against the toxicity of tertiary-butyl
hydroperoxide is the enzyme glutathione peroxidase, which relies on glutathione for
reducing equivalents, we reason that the depletion of glutathione is responsible for the
enhanced cytotoxicity observed with metallothionein induction. Not all of the effect of
dexamethasone and interleukin-6 treatment on glutathione was dependent on
metallothionein expression, however, since glutathione was also reduced to some extent
by this treatment in cultures from KO mice as well.

At least two hypotheses have been generated to explain this phenomenon (Fig. 6-
2). The first hypothesis (the author's) is that metallothionein induction depletes the
available cellular cysteine pools. Evidence for this includes the fact that cysteine residues
make up 1/3 of the total amino acid residues of both metallothionein and glutathione.
Also, the absolute cysteine content of the metallothionein pool (after induction of
metallothionein by the various treatments used in this experiment) is comparable to the
cysteine content of the glutathione pool. Further, the glutathione content of primary
hepatocyte cultures is highly dependent on the available cysteine content of the culture
medium (Wang et al. 1997).

The second hypothesis (of Dr. Cousins) is that the metallothionein induced by
various treatments physically interacts with glutathione, and thereby interferes with the

Cysteine

BOH 4 W GSSgBB !^^ NAD

^GSH Px r w 'GSH Rx

TBOOH

NADP

Figure 6-2. Mechanisms by which metallothionein might cause glutathione depletion.
Metallothionein might interfere with glutathione regeneration through physical
interactions with oxidized glutathione. Alternatively, induction of metallothionein
might consume enough cysteine so as to interfere with glutathione synthesis.

use and/or availablility of glutathione as a substrate for glutathione peroxidase. Evidence
for this viewpoint includes the fact that glutathione forms mixed disulfides with a number
of intracellular sulfhydryl-containing proteins, as well as the fact that several putative
glutathione binding sites have been reported in the metallothionein molecule (Brouwer et
al. 1993, Maret 2000). This hypothesis includes observations from in vitro experiments
where glutathione peroxidase and reduced glutathione release zinc from metallothionein
and cellular oxidants cause release of cellular glutathione (oxidized and reduced). There
is no reason to believe that the mechanisms outlined in these hypotheses would be
mutually exclusive. If metallothionein does affect hepatic glutathione levels in vivo, this
relationship might explain hepatic glutathione depression seen after treatment with a
certain hormones, or under some stress conditions.

In summary, the findings of these studies agree with the previously proposed
theories regarding the role of metallothionein in zinc metabolism. The results herein
refine this model by constraining it to conditions of highly excessive zinc intake. A more
novel finding was that metallothionein may regulate its own expression by providing a
labile intracellular zinc pool that may feedback positively on metallothionein gene
expression.

Metallothionein expression protected against oxidative stress in vivo, but
metallothionein overexpression and supplemental dietary zinc provided no further
protection. We interpret this to mean that there is a threshold of zinc intake and
metallothionein expression that is protective, but above which is not helpful. Normal
metallothionein expression combined with the required zinc intake meet this threshold
value in mice. These results are not consistent with direct antioxidant roles for

metallothionein or zinc. The results may be more reflective of the need for appropriate
regulation of zinc metabolism during oxidative stress, since zinc metabolism was highly
altered in KO mice. Further, studies in primary hepatocyte cultures suggest that
glutathione depletion may result in cells that experience metallothionein overexpression,
and that glutathione depletion may compromise oxidant defenses.

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BIOGRAPHICAL SKETCH

Steven Roger Davis was born on August 3, 1968 in Worcester, Massachusetts. He
was raised in Sutton, Massachusetts and graduated from Sutton High School in 1986. He
received his Bachelor of Science degree in Chemistry at Worcester State College in
Worcester, Massachusetts in 1992. He then worked as a chemistry laboratory
instructor/supervisor for two years at the College of the Holy Cross in Worcester,
Massachusetts before coming to the University of Florida to pursue a doctorate in
Nutritional Sciences.

109

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quantity, as a dissertation for the degree of Doctor of Philosophy

Robert J. Cousins, Chair
Boston Family Professor of
Human Nutrition

5esse F. Gregory, III \U -J
Professor of Food Science and
Human Nutrition

Susan S. Percival

Associate Professor of Food Science
and Human Nutrition

*:*zLS

Rachel B. Shireman
Professor of Food Science and
Human Nutrition

Stephen M. Roberts

Professor of Veterinary Medicine

This dissertation was submitted to the Graduate Faculty of the College of Agricultural
and Life Sciences and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.

December 2000 T^'

«srQ_

Dean, College of Agricultural an
Life Sciences

Dean, Graduate School

LD
1780

20 M

UNIVERSITY OF FLORIDA

3 1262 08555 3658

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